Accessibility / Report Error

Does low-intensity pulsed ultrasound accelerate phasic calcium phosphate ceramic-induced bone formation?

ABSTRACT

Purpose:

Low-intensity pulsed ultrasound (LIPUS) has been used to stimulate the healing of the fresh fracture, delayed union, and non-union in both animal and clinical studies. Besides, biphasic calcium phosphate ceramic (BCP) is a promising biomaterial for bone repair as it shows favorable biocompatibility, osteoinduction, and osteoconduction. However, scarcity is known about the combined effect of LIPUS and BCP on bone formation.

Methods:

The combined effect of LIPUS and BCP was studied in a beagle model. Twelve dogs were used. BCP granules without any additions were implanted into bilateral erector spinae muscles. One side is the BCP group, while the counterlateral side is LIPUS + BCP group. Histological and histomorphometric analyses, and quantitative real-time polymerase chain reaction were evaluated.

Results:

Compared with BCP alone, the LIPUS + BCP showed no advantages in early bone formation. Furthermore, the Notch signaling pathway-related mRNA has no significant difference between the two groups.

Conclusions:

The preliminary results showed that the BCP, which has intrinsic osteoinduction nature, was an effective and promising material. However, LIPUS has no enhanced effect in BCP induced ectopic bone formation. Furthermore, LIPUS has no effect on the Notch signaling pathway. Whether costly LIPUS could be used in combination with BCP should be a rethink.

Key words
Ultrasonic Waves; Osteogenesis; Biocompatible Materials

Introduction

Critical-sized bone defects and non-unions requiring bone grafting are still challenges in clinical routine, as they could prolong the length of hospitalization and increase health-care expenditure. Commonly, natural bone grafts and synthetic biomaterials are used in such a scenario. However, the use of natural bone grafts is limited by the intrinsic disadvantages of donor site damage, the limited amount of bone mass (autografts), immunogenicity, and disease transmission (xenografts)11 Baldwin P, Li DJ, Auston DA, Mir HS, Yoon RS, Koval KJ. Allograft, and bone graft substitutes: clinical evidence and indications for use in the setting of orthopaedic trauma surgery. J Orthop Trauma. 2019;33(4):203-13. https://doi.org/10.1097/BOT.0000000000001420
https://doi.org/10.1097/BOT.000000000000...
. Therefore, the exploration of effective biomaterials is imperative for clinical use. Among the plethora of available biomaterials, biphasic calcium phosphate ceramics showed satisfactory efficacy and reached clinical use22 Ho-Shui-Ling A, Bolander J, Rustom LE, Johnson AW, Luyten FP, PIcart C. Bone regeneration strategies: engineered scaffolds, bioactive molecules and stem cells current stage and future perspectives. Biomaterials. 2018;180:143-62. https://doi.org/10.1016/j.biomaterials.2018.07.017
https://doi.org/10.1016/j.biomaterials.2...
. However, the efficiency and mechanical strength of biomaterials-induced bone formation are still far from satisfactory.

Except for bone graft, several auxiliary therapies have also been studied to accelerating bone regeneration. Among them, low-intensity pulsed ultrasound (LIPUS) has drawn increasing attention because of its non-invasive physical stimulation for therapeutic applications. Several animal studies33 Naruse K, Uchino M, Hirakawa N, Toyama M, Miyajima G, Mukai M, Urabe K, Uchida K, Itoman M. 4. The Low-Intensity Pulsed Ultrasound (LIPUS) mechanism and the effect of teriparatide on fracture healing. J Orthop Trauma. 2016;30(8):S3. https://doi.org/10.1097/01.bot.0000489989.28108.21
https://doi.org/10.1097/01.bot.000048998...

4 Katano M, Naruse K, Uchida K, Mikuni-Takagaki Y, Takaso M, Itoman M, Urabe K. Low intensity pulsed ultrasound accelerates delayed healing process by reducing the time required for the completion of endochondral ossification in the aged mouse femur fracture model. Exp Anim. 2011;60(4):385-95. https://doi.org/10.1538/expanim.60.385
https://doi.org/10.1538/expanim.60.385...

5 Naruse K, Sekiya H, Harada Y, Iwabuchi S, Kozai Y, Kawamata R, Kashima I, Uchida K, Seto K, Itoman K, Mikuni-Takagaki Y. Prolonged endochondral bone healing in senescence is shortened by low-intensity pulsed ultrasound in a manner dependent on COX-2. Ultrasound Med Biol. 2010;36(7):1098-108. https://doi.org/10.1016/j.ultrasmedbio.2010.04.011
https://doi.org/10.1016/j.ultrasmedbio.2...
-66 Azuma Y, Ito M, Harada Y, Takagi H, Ohta T, Jingushi S. Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus. J Bone Miner Res. 2001;16(4):671-80. https://doi.org/10.1359/jbmr.2001.16.4.671
https://doi.org/10.1359/jbmr.2001.16.4.6...
proved positive effects of LIPUS for promoting faster healing of femoral fracture in aged or osteoporotic rats. A similar healing effect was also confirmed on sheep and rabbit models77 Mayr E, Laule A, Suger G, Ruter A, Claes L. Radiographic results of callus distraction aided by pulsed low-intensity ultrasound. J Orthop Trauma. 2001;15(6):407-14. https://doi.org/10.1097/00005131-200108000-00005
https://doi.org/10.1097/00005131-2001080...
,88 Pilla AA, Mont MA, Nasser PR, Khan SA, Figueiredo M, Kaufman JJ, Siffert RS. Non-invasive low-intensity pulsed ultrasound accelerates bone healing in the rabbit. J Orthop Trauma. 1990;4(3):246-53. https://doi.org/10.1097/00005131-199004030-00002
https://doi.org/10.1097/00005131-1990040...
.

The combined effect of LIPUS with ceramic biomaterials has also drawn attention in the recent one decade. Researchers studied whether LIPUS could accelerate ceramic biomaterials-induced bone formation. Wu et al.99 Wu L, Lin L, Qin YX. Enhancement of cell ingrowth, proliferation, and early differentiation in a three-dimensional silicon carbide scaffold using low-intensity pulsed ultrasound. Tissue Eng Part A. 2014;21(1-2):53-61. https://doi.org/10.1089/ten.TEA.2013.0597
https://doi.org/10.1089/ten.TEA.2013.059...
proved in vitro that LIPUS facilitated the ingrowth, proliferation, and early differentiation of MC3T3-E1 mouse preosteoblasts in silicon carbide porous ceramic scaffold. Iwai et al.1010 Iwai T, Harada Y, Imura K, Iwabuchi S, Murai J, Hiramatsu K, Myoui A, Yoshikawa H, Tsumaki N. Low-intensity pulsed ultrasound increases bone ingrowth into porous hydroxyapatite ceramic. J Bone Miner Metab. 2007;25(6):392-9. https://doi.org/10.1007/s00774-007-0777-5
https://doi.org/10.1007/s00774-007-0777-...
applied LIPUS to porous hydroxyapatite ceramic implanted in the femoral condyle of rabbits and detected significantly increased osteoblast number and mineralized volume. However, whether LIPUS could facilitate BCP-induced bone formation is still unclear. Thus, we designed an experimental beagle model to evaluate the effectiveness of the combination of LIPUS and BCP as a valuable combinatorial tool for clinical use.

Methods

Materials

BCP ceramics granules (Φ 5 mm × 8 mm) were purchased from the National Engineering Research Center for Biomaterials (Chengdu, Sichuan, People’s Republic of China). 60wt.% hydroxyapatite and 40wt.% β-tricalcium phosphates were prepared using wet precipitation and hydrogen peroxide (H2O2) foaming method and then sintered for 3 hours at 1,100 °C. The macropore size was 380 ± 50 μm, overall porosity (%vol) was 70.8%, and specific weight was 0.970 g/cm3 (Table 1)1111 Guo X, Jiang H, Zong X, Du L, Zhao J, Zhang D, Song G, Jin X. The implication of the notch signaling pathway in biphasic calcium phosphate ceramic-induced ectopic bone formation: a preliminary experiment. J Biomed Mater Res A. 2020;108(5):1035-44. https://doi.org/10.1002/jbm.a.36878
https://doi.org/10.1002/jbm.a.36878...
. The morphology was analyzed by scanning electron microscope (SEM, Philips XL20, Dutch).

Table 1
Relevant parameters of biphasic calcium phosphate.

Animal model

The experiment was conducted using a self-control method. Twelve beagle dogs (10 months old, 10-12 kg, half male and half female) were used. General anesthesia was achieved by abdominal injection sodium pentobarbital (30 mg/kg). The animals were placed in the prone position, and the back was shaved. After sterilizing, an 8-cm longitudinal incision was made in the dorsum, four intramuscular pockets were made, and eight BCP granules were inserted into one pocket. The wound was closed layer by layer (Fig. 1). Penicillin was intramuscularly injected consecutively during the first three days after surgery to prevent infections. The present study was approved by the Institutional Animal Care and Use Committee of our hospital. Animal experiments were carried out following the National Institutes of Health guide for the care and use of laboratory animals.

Figure 1
(a) Low-intensity pulsed ultrasound device; (b and c) the gross scanning electron microscopegraphs of the biphasic calcium phosphategranule; (d) the gross graph of the excavated specimen.

Low-intensity pulsed ultrasound treatment

The LIPUS device (Osteoron IV, Japan) was daily used on the right side of the operated back. The apparatus was set at 1 KHz frequency, 0.3 W/cm2 intensity, duty cycle of 20% for 2 0min. After four, eight and 12 weeks, four dogs were sacrificed randomized by an overdose of sodium pentobarbital, and the implants were harvested (Fig. 1a).

Histological evaluation of neo-ossification

Excavated samples were halved for both histological and quantitative real-time polymerase chain reaction (qRT-PCR) study. Half of the samples were fixed in 4% paraformaldehyde, and then decalcified using 10% ethylene diamine tetraacetic for one~two weeks, and after dehydrated in ascending concentrations of ethanol and embedded in paraffin. Tissue sections were sliced, and the cross section was stained with hematoxylin/eosin (HE) staining. Occurrence of bone or osteoid tissue formation was assessed by one laboratory technician and one experienced pathologist, who were unknown to the assignment of groups. Histomorphometry was performed according to previous report1111 Guo X, Jiang H, Zong X, Du L, Zhao J, Zhang D, Song G, Jin X. The implication of the notch signaling pathway in biphasic calcium phosphate ceramic-induced ectopic bone formation: a preliminary experiment. J Biomed Mater Res A. 2020;108(5):1035-44. https://doi.org/10.1002/jbm.a.36878
https://doi.org/10.1002/jbm.a.36878...

12 Manchón A, Alkhraisat M, Rueda-Rodriguez C, Torres J, Prados-Frutos JC, Ewald A, Gbureck U, Cabrejos-Azama J, Rodriguez-Gonzalez A, Lopez-Cabarcos E. Silicon calcium phosphate ceramic as novel biomaterial to simulate the bone regenerative properties of autologous bone. J Biomed Mater Res A. 2015;103(2):479-88. https://doi.org/10.1002/jbm.a.35196
-1313 Wang L, Zhang B, Bao C, Habibovic P, Hu J, Zhang X. Ectopic osteoid and bone formation by three calcium-phosphate ceramics in rats, rabbits and dogs. PLoS One. 2014;9(9):e107044. https://doi.org/10.1371/journal.pone.0107044
https://doi.org/10.1371/journal.pone.010...
. Incidence of neo-ossification (In) was defined as bone/osteoid filled pores in randomly selected non-overlapping fields at a magnification of 5× (Eq. 1).

P 1   =   the   number   of   bone   and   osteoid   filled   pores   /   the   number   of   overall   pores (1)

Pores which were partially exhibited in one figure were not calculated.

Volume of neo-ossification (Vn) was defined as the percentage of bone/osteoid tissue inside the pores (P2) in 10× magnified fields (Eq. 2).

Vn   =   ( bone   and   osteoid   area   within   pore   1 # / area   of   the   pore   1 #   + bone   and   osteoid   area   within   pore   2 # / area   of   the   pore   2 #   + . . . +   bone   and   osteoid   area   within   pore   n # / area   of   the   pore   n # ) / n (2)

n: the number of intact pores in every 10× magnified field.

The area of the pores and the bone/osteoid area inside the pores were measured respectively using ImageJ software.

Quantitative real-time polymerase chain reaction

After implantation for four and eight weeks, BCP granules were excavated and stored in RNAlater Stabilization Solution (Thermofisher, Waltham, United States of America). The central part of the sample was used for RNA isolation using the RNeasy kit (Qiagen, Venlo, Netherlands). Complementary DNA (cDNA) was obtained by reverse transcription of 1,000 ng of total RNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Transgen, China). qRT-PCR was performed on a light cycler PCR machine (Roche, Switzerland) using SYBR green I master mix (Roche, Switzerland) three times. The following thermocycling conditions were used: initial denaturation at 95 °C for 10 minutes; followed by 40 amplification cycles (denaturation at 95 °C for 15 sec, annealing at 60 °C for 60 sec and extension at 72 °C for 45 sec).

As showed in Table 2, the primer sequences were referred to previous published articles or designed using the Primer 6 software (Hewlett-Packard, Madrid, Spain). The expression of osteogenesis and the Notch signaling pathway-related genes was normalized to GAPDH levels. The data were analyzed using the 2-AACq method (25) and LightCycler® 96 software version 1.1 (Roche Diagnostics GmbH). The experiment was performed in triplicate, and the results are expressed as relative fold changes.

Table 2
Primers for the quantitative real-time polymer chain reaction.

Statistics

Light micrograph images were analyzed using ImageJ software, and the result was reported as mean±standard deviation (SD). The statistical analysis was calculated using SPSS 20.0 software (SPSS, Inc., California, United States of America), and variation between samples was analyzed using paired-t test if the numbers are normally distributed. If not, the Wilcoxon signed-rank test was used. p < 0.05 was considered as significant difference.

Results

Gross and scanning electron microscope graphs showed structures of biphasic calcium phosphate granules

The BCP were porous with similar macropores and micropores. The surface of BCP exhibited roughness, and adjacent macropores was connected. The macroporosity and interconnectivity facilitate colonization of cells and perfusion of nutrition (Figs. 1b and 1c).

Gross evaluation of explants

No surgical complications including infection/wound dehiscence were detected. Under compression of erector spinae muscle, the majority of implanted BCP granules were accumulated in a cylinder shape and surrounded by muscular and fibrous connective tissue (Fig. 1d). At four weeks, although connected, the BCP particles were intact and could be easily separated. At eight weeks, the BCP particles were incorporated with fibrous tissue and could be easily sliced down after one-week decalcification. After 12 weeks, the BCP particles were surrounded by thick fibrous capsule and need at least two-week decalcification before histologic analysis. The volume of samples remained unchanged, with no significant macroscopic increase or decrease.

Histomorphometrical evaluation of neo-ossification

Figure 2 showed the overview of bone or osteoid tissue formation at four, eight and 12 weeks after implantation. Bone or osteoid tissue was scarcely observed at four weeks, only fibroblast-like, monocyte-like could be seen. At eight weeks, bone or osteoid tissue was stained in red as it was acidophilia. Newly formed bone or osteoid tissue was in the peripheral areas, while fibrous tissue and neovascularization dwelled in the central area. Furthermore, fibrous tissue and neovascularized tissue also existed in the connection between macropores.

Figure 2
Hematoxylin/eosin staining of the biphasic calcium phosphate granules. (a-c) Biphasic calcium phosphate group; (d-f) biphasic calcium phosphate + low-intensity pulsed ultrasound group. The left column was four weeks, the middle column was eight weeks,while the right column was twelve weeks.

Figure 3a depicted the incidence of bone or osteoid tissue in BCP and BCP + LIPUS groups. At four and eight weeks, BCP group showed a slightly higher incidence of bone or osteoid tissue when compared with LIPUS additional group. However, the difference was not statistically significant.

Figure 3
The histomorphometrical result of neo-ossification. (a) The incidence of bone or osteoid tissue. (b) The volume of the bone or osteoid tissue in macropores.

Figure 3b illustrated the volume of bone or osteoid tissue in BCP and BCP + LIPUS groups. Quantification of % of BCP-induced ectopic bone formation in macropores showed that, at the fourth and eighth weeks, there was no difference in BCP and BCP + LIPUS groups. At the 12th week, the volume of neo-ossification in BCP group was higher than in the BCP + LIPUS group.

Quantitative real-time polymerase chain reaction of osteogenesis-related genes

Alkaline phosphatase (ALP), osteocalcin (OC), and osteopontin (OPN) were selected as classic osteogenesis-related genes. At the fourth week, both BCP and BCP + LIPUS groups expressed the lowest level in all genes. At the eighth week, the mRNA expression in the targeted genes increased parallelly. However, for ALP and OC mRNA, no differences were detected between groups. The LIPUS might add positive effect in increasing the mRNA expression of OPN (Fig. 4).

Figure 4
The expression of mRNA of osteogenesis and the Notch signaling pathway-related genes.

Quantitative real-time polymerase chain reaction of Notch signaling pathway-related genes

Guo et al.1111 Guo X, Jiang H, Zong X, Du L, Zhao J, Zhang D, Song G, Jin X. The implication of the notch signaling pathway in biphasic calcium phosphate ceramic-induced ectopic bone formation: a preliminary experiment. J Biomed Mater Res A. 2020;108(5):1035-44. https://doi.org/10.1002/jbm.a.36878
https://doi.org/10.1002/jbm.a.36878...
conducted a preliminary experiment and found the pivotal role of Notch signaling pathway in BCP-induced ectopic bone formation. Thus, in the present study, Notch signaling pathway-related genes were also evaluated. There were no differences in Jagged1/hey1 mRNA expression between BCP and BCP + LIPUS groups. Runt related transcription factor 2 (RUNX2) is a critical factor in the regulation of osteogenic differentiation. BCP + LIPUS showed decreased expression of RUNX2 mRNA (Fig. 4).

Discussion

LIPUS is a therapeutical ultrasound which can be transmitted as acoustical pressure waves in tissues. This low-intensity ultrasound is non-thermogenic, without destruction in biological tissue1414 Jiang X, Savchenko O, Li Y, Qi S, Yang T, Zhang W, Chen J. A review of low-intensity pulsed ultrasound for therapeutic applications. IEEE Trans Biomed Eng. 2019;66(10):2704-18. https://doi.org/10.1109/TBME.2018.2889669
https://doi.org/10.1109/TBME.2018.288966...
. In 1994 and 2010, United States of America and United Kingdom separately approved the clinical use of LIPUS for fresh fracture healing and non-unions. From then on, because miscellaneous experimental studies supported the positive effect of LIPUS in accelerating bone healing1515 Leung KS, Lee WS, Tsui HF, Liu PP, Cheung W. Complex tibial fracture outcomes following treatment with low-intensity pulsed ultrasound. Ultrasound Med Biol. 2004;30(3):389-95. https://doi.org/10.1016/j.ultrasmedbio.2003.11.008
https://doi.org/10.1016/j.ultrasmedbio.2...
, cartilage repair1616 Yang SW, Kuo CL, Chang SJ, Lin YT, Manousakas I, Kuo SM. Does low-intensity pulsed ultrasound treatment repair articular cartilage injury? A rabbit model study. BMC Musculoskelet Disord. 2014;15:36. https://doi.org/10.1186/1471-2474-15-36
https://doi.org/10.1186/1471-2474-15-36...
,1717 Zhou HY, Li Q, Wang JX, Xie YJ, Wang SQ, Lei L, Gao YQ, Huang MM, Hu Y, Xu FY, Zhang C. Low-intensity pulsed ultrasound repair in mandibular condylar cartilage injury rabbit model. Arch Oral Biol. 2019;104:60-6. https://doi.org/10.1016/j.archoralbio.2019.05.018
https://doi.org/10.1016/j.archoralbio.20...
, and even in neuron growth1818 Zhao L, Feng Y, Hu H, Shi A, Zhang L, Wan M. Low-intensity pulsed ultrasound enhances nerve growth factor-induced neurite outgrowth through mechanotransduction-mediated ERK1/2-CREB-Trx-1 signaling. Ultrasound Med Biol. 2016;42(12):2914-25. https://doi.org/10.1016/j.ultrasmedbio.2016.07.017
https://doi.org/10.1016/j.ultrasmedbio.2...
and erectile dysfunction1919 Cui W, Li H, Guan R, Li M, Yang B, Xu Z, Lin M, Tian L, Zhang X, Li B, Liu W, Dong Z, Wang Z, Zheng T, Zhang W, Lin G, Guo Y, Xin Z. Efficacy and safety of novel low-intensity pulsed ultrasound (LIPUS) in treating mild to moderate erectile dysfunction: a multicenter, randomized, double-blind, sham-controlled clinical study. Transl Androl Urol. 2019;8(4):307-19. https://doi.org/10.21037/tau.2019.07.03
https://doi.org/10.21037/tau.2019.07.03...
, LIPUS has become a highly recommended device with an increasing scope of clinical application.

In clinical trials, several studies proved that LIPUS enhanced healing rate among fresh fractures and nonunion > one year1515 Leung KS, Lee WS, Tsui HF, Liu PP, Cheung W. Complex tibial fracture outcomes following treatment with low-intensity pulsed ultrasound. Ultrasound Med Biol. 2004;30(3):389-95. https://doi.org/10.1016/j.ultrasmedbio.2003.11.008
https://doi.org/10.1016/j.ultrasmedbio.2...
,2020 Zura R, Della Rocca GJ, Mehta S, Harrison A, Brodie C, Jones J, Steen RG. Treatment of chronic (>1 year) fracture nonunion: heal rate in a cohort of 767 patients treated with low-intensity pulsed ultrasound (LIPUS). Injury. 2015;46(10):2036-41. https://doi.org/10.1016/j.injury.2015.05.042
https://doi.org/10.1016/j.injury.2015.05...
,2121 Lou S, Lv H, Li Z, Tang P, Wang Y. Effect of low-intensity pulsed ultrasound on distraction osteogenesis: a systematic review and meta-analysis of randomized controlled trials. J Orthop Surg Res. 2018;13(1):205. https://doi.org/10.1186/s13018-018-0907-x
https://doi.org/10.1186/s13018-018-0907-...
. Moreover, compared with the whole population, older patients (≥ 60) with fracture risk factors treated with LIPUS had similar heal rates2222 Zura R, Mehta S, Della Rocca GJ, Jones J, Steen RG. A cohort study of 4,190 patients treated with low-intensity pulsed ultrasound (LIPUS): findings in the elderly versus all patients. BMC Musculoskelet Disord. 2015;16:45. https://doi.org/10.1186/s12891-015-0498-1
https://doi.org/10.1186/s12891-015-0498-...
. However, more and more clinical investigations are contentious with contradictory results. Hannemann et al.2323 Hannemann PF, Mommers EH, Schots JP, Schots JP, Brink PR, Poeze M. The effects of low-intensity pulsed ultrasound and pulsed electromagnetic fields bone growth stimulation in acute fractures: a systematic review and meta-analysis of randomized controlled trials. Arch Orthop Trauma Surg. 2014;134(8):1093-106. https://doi.org/10.1007/s00402-014-2014-8
https://doi.org/10.1007/s00402-014-2014-...
concluded that, although LIPUS accelerated bone healing when considering the time to radiological and clinical union, current evidence from randomized clinical trials was inadequate to support the benefit of LIPUS in reducing the incidence of nonunions for acute fracture treatment. Lou et al.2121 Lou S, Lv H, Li Z, Tang P, Wang Y. Effect of low-intensity pulsed ultrasound on distraction osteogenesis: a systematic review and meta-analysis of randomized controlled trials. J Orthop Surg Res. 2018;13(1):205. https://doi.org/10.1186/s13018-018-0907-x
https://doi.org/10.1186/s13018-018-0907-...
conducted a meta-analysis regarding the effect of LIPUS on distraction osteogenesis and found no beneficial effect of LIPUS in reducing treatment time or complication rate. When it comes to non-unions, Leighton et al.2424 Leighton R, Watson JT, Giannoudis P, Papakostidis C, Harrison A, Steen RG. Healing of fracture nonunions treated with low-intensity pulsed ultrasound (LIPUS): a systematic review and meta-analysis. Injury. 2017;48(7):1339-47. https://doi.org/10.1016/j.injury.2017.05.016
https://doi.org/10.1016/j.injury.2017.05...
summarized that LIPUS could benefit patients for whom surgery is high risk due to no spontaneous healing of definite fracture non-unions was expected. Thus, LIPUS is recommended alternative to surgery for this scenario.

Increasingly updated studies question the traditional findings. Busse et al.2525 Busse JW, Bhandari M, Einhorn TA, Schemitsch E, Heckman JD, 3rd RT, Leung KS, Heels-Ansdell D, Makosso-Kallyth S, Rocca GJD, Jones CB, Guyatt GH. Re-evaluation of low intensity pulsed ultrasound in treatment of tibial fractures (TRUST): randomized clinical trial. BMJ. 2016;355:i5351. https://doi.org/10.1136/bmj.i5351
https://doi.org/10.1136/bmj.i5351...
conducted a multi-center RCT across North American academic trauma centers and concluded that LIPUS after tibial fracture fixation did not accelerate neither radiographic healing nor functional recovery. Furthermore, a clinical practice guideline published in 2017 issued a strong recommendation against LIPUS in fresh fracture or osteotomy and even non-unions.

In the present study, the gross observation and histomorphometrical calculation of ectopic bone formation showed no beneficial effect of LIPUS on BCP-induced ectopic bone formation. Furthermore, osteogenesis-related genes ALP/OC showed no difference between LIPUS + BCP and BCP groups. Although the mRNA expression of OPN was increased in the LIPUS + BCP group, the change was trivial. The synthesized results were insufficient to conclude a benefit of the combination of LIPUS and BCP as a combinatorial tool for clinical use. The finding is different from previous experiment by Fan et al.2626 Fan B, Guo Z, Li X, Li S, Gao P, Xiao X, Wu J, Shen C, Jiao Y, Hou W. Electroactive barium titanate coated titanium scaffold improves osteogenesis and osseointegration with low-intensity pulsed ultrasound for large segmental bone defects. Bioact Mater. 2020;5(4):1087-101. https://doi.org/10.1016/j.bioactmat.2020.07.001
https://doi.org/10.1016/j.bioactmat.2020...
. They studied the combined effect of LIPUS and porous BaTiO3/pTi scaffold and found that the adhesion, proliferation, gene expression of bone marrow derived stem cell were increased by LIPUS in vitro, and osteogenesis/osseointegration was promoted in large segmental defect repair of rabbit model.

The inefficiency of LIPUS in BCP-induced ectopic bone formation and the disparity of present and previous studies may be due to the difference of biomaterial and animal model. First, the similar mechanism may explain why LIPUS showed no adjuvant effect to BCP-induced bone formation. In the present study, BCP was used as porous scaffold. The mechanism of BCP and LIPUS-induced bone regeneration is overlapped. Several proposed theories of BCP-induced de new osteogenesis are:

  • Ca2+ and PO43- ions released from BCP intrigue bone formation;

  • Several growth factors including BMPs (Bone Morphogenetic Protein)/VEGF (Vascular Endotelial Growth Factor) absorbed by the porous ceramic promote bone formation and neovascularization;

  • Surface structure physically stimulates cellular signaling pathway and thereby intrigues osteogenesis2727 Xiao D, Zhang J, Zhang C, Barbieri D, Yuan H, Moroni L, Feng G. The role of calcium phosphate surface structure in osteogenesis and the mechanisms involved. Acta Biomater. 2020;106:22-33. https://doi.org/10.1016/j.actbio.2019.12.034
    https://doi.org/10.1016/j.actbio.2019.12...
    ,2828 Ebrahimi M, Botelho M. Biphasic calcium phosphates (BCP) of hydroxyapatite (HA) and tricalcium phosphate (TCP) as bone substitutes: importance of physicochemical characterizations in biomaterials studies. Data Brief. 2017;10:93-7. https://doi.org/10.1016/j.dib.2016.11.080
    https://doi.org/10.1016/j.dib.2016.11.08...
    .

Similarly, the mechanism of LIPUS-induced bone regeneration is also associated with ions uptake and angiogenesis. Tassinary et al.2929 Tassinary JAF, Lunardelli A, Basso BS, Dias HB, Catarina AV, Stulp S, Haute GV, Martha BA, Melo DAS, Nunes FB, Donardio MVF, Oliveira JR. Low-intensity pulsed ultrasound (LIPUS) stimulates mineralization of MC3T3-E1 cells through calcium and phosphate uptake. Ultrasonics. 2018;84:290-5. https://doi.org/10.1016/j.ultras.2017.11.011
https://doi.org/10.1016/j.ultras.2017.11...
proved that LIPUS was capable of stimulating differentiation and mineralization of MC3T3-E1 cells through Ca2+ and PO43- uptake. Meanwhile, LIPUS can increase the mRNA level of vascular endothelial growth factor A2626 Fan B, Guo Z, Li X, Li S, Gao P, Xiao X, Wu J, Shen C, Jiao Y, Hou W. Electroactive barium titanate coated titanium scaffold improves osteogenesis and osseointegration with low-intensity pulsed ultrasound for large segmental bone defects. Bioact Mater. 2020;5(4):1087-101. https://doi.org/10.1016/j.bioactmat.2020.07.001
https://doi.org/10.1016/j.bioactmat.2020...
. Secondly, the ectopic bone formation model did not put the roles LIPUS exerted on the residual bone defect to good use. In the present study, ectopic bone formation rather than in-situ bone repair model was used. In in-situ repair model, biomaterials exert osteoinductive and osteoconductive function, while merely exerting osteoinductive effect in ectopic bone formation3030 Winkler T, Sass FA, Duda GN, Schmidt-Beek K. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: the unsolved challenge. Bone Joint Res. 2018;7(3):232-43. https://doi.org/10.1302/2046-3758.73.BJR-2017-0270.R1
https://doi.org/10.1302/2046-3758.73.BJR...
. The biomechanical waves of LIPUS on residual bone defect can produce additional stimulation and promote bone regeneration. Thus, it may put both the biomaterial-induced bone integration/bone regeneration and residual bone-induced bone regeneration to good use. Thirdly, LIPUS does not always showed an additional effect to implantable materials. Similarly, Ishihara et al.3131 Ishihara Y, Ueki K, Sotobori M, Marukawa K, Moroi A. Bone regeneration by statin and low-intensity pulsed ultrasound (LIPUS) in rabbit nasal bone. J Craniomaxillofac Surg. 2014;42(3):185-93. https://doi.org/10.1016/j.jcms.2013.05.002
https://doi.org/10.1016/j.jcms.2013.05.0...
studied the combined effect of LIPUS with statin in nasal bone defect and found not statistical difference between combined group from LIPUS alone or statin alone.

Notch is an important signaling pathway in bone repair and regeneration. Guo et al.1111 Guo X, Jiang H, Zong X, Du L, Zhao J, Zhang D, Song G, Jin X. The implication of the notch signaling pathway in biphasic calcium phosphate ceramic-induced ectopic bone formation: a preliminary experiment. J Biomed Mater Res A. 2020;108(5):1035-44. https://doi.org/10.1002/jbm.a.36878
https://doi.org/10.1002/jbm.a.36878...
reported that Notch signaling pathway also regulated in BCP-induced ectopic bone formation. In the present study, Notch signaling pathway related genes, Jagged1 and Hey 1 showed no change in the LIPUS + BCP group. The mRNA of RUNX2 even reduced in the LIPUS + BCP group. RUNX2 is also a pivotal downstream transcription factor which could be influenced by several signaling pathways, including Notch signaling pathway, BMP signaling pathway, Wnt signaling pathway. Contrary with previous study, BCP + LIPUS showed decreased expression of RUNX2 mRNA, which indicated the negative effect of LIPUS in BCP-induced ectopic bone formation.

The present experiment has several limitations. First, only ectopic bone formation was used. Further study of in-situ bone defect model is warranted to study the combinatorial effect of LIPUS and BCP. Second, the sample size was relatively small. Third, only one apparatus and one kind of BCP was studied on the present study.

Conclusions

BCP, which has intrinsic osteoinduction nature, was an effective and promising material. However, LIPUS has no enhanced effect in BCP-induced ectopic bone formation. Furthermore, LIPUS has no effect with the Notch signaling pathway. Whether costly LIPUS could be used in combination with BCP should be rethink.

Acknowledgments

To Dong Zhang for his help in the animal experiment.

  • Data availability statement

    Data will be available upon request.
  • Funding

    Key Project of Dean’s Research Assistance Fund of Stomatology Hospital of Jinan
    Grant no. 2018-01
    Fundamental Research Funds for the Central Universities
    Grant no. 3332022034
    Medical and Health Science Development Project
    Grant no. 2019WS072

References

  • 1
    Baldwin P, Li DJ, Auston DA, Mir HS, Yoon RS, Koval KJ. Allograft, and bone graft substitutes: clinical evidence and indications for use in the setting of orthopaedic trauma surgery. J Orthop Trauma. 2019;33(4):203-13. https://doi.org/10.1097/BOT.0000000000001420
    » https://doi.org/10.1097/BOT.0000000000001420
  • 2
    Ho-Shui-Ling A, Bolander J, Rustom LE, Johnson AW, Luyten FP, PIcart C. Bone regeneration strategies: engineered scaffolds, bioactive molecules and stem cells current stage and future perspectives. Biomaterials. 2018;180:143-62. https://doi.org/10.1016/j.biomaterials.2018.07.017
    » https://doi.org/10.1016/j.biomaterials.2018.07.017
  • 3
    Naruse K, Uchino M, Hirakawa N, Toyama M, Miyajima G, Mukai M, Urabe K, Uchida K, Itoman M. 4. The Low-Intensity Pulsed Ultrasound (LIPUS) mechanism and the effect of teriparatide on fracture healing. J Orthop Trauma. 2016;30(8):S3. https://doi.org/10.1097/01.bot.0000489989.28108.21
    » https://doi.org/10.1097/01.bot.0000489989.28108.21
  • 4
    Katano M, Naruse K, Uchida K, Mikuni-Takagaki Y, Takaso M, Itoman M, Urabe K. Low intensity pulsed ultrasound accelerates delayed healing process by reducing the time required for the completion of endochondral ossification in the aged mouse femur fracture model. Exp Anim. 2011;60(4):385-95. https://doi.org/10.1538/expanim.60.385
    » https://doi.org/10.1538/expanim.60.385
  • 5
    Naruse K, Sekiya H, Harada Y, Iwabuchi S, Kozai Y, Kawamata R, Kashima I, Uchida K, Seto K, Itoman K, Mikuni-Takagaki Y. Prolonged endochondral bone healing in senescence is shortened by low-intensity pulsed ultrasound in a manner dependent on COX-2. Ultrasound Med Biol. 2010;36(7):1098-108. https://doi.org/10.1016/j.ultrasmedbio.2010.04.011
    » https://doi.org/10.1016/j.ultrasmedbio.2010.04.011
  • 6
    Azuma Y, Ito M, Harada Y, Takagi H, Ohta T, Jingushi S. Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus. J Bone Miner Res. 2001;16(4):671-80. https://doi.org/10.1359/jbmr.2001.16.4.671
    » https://doi.org/10.1359/jbmr.2001.16.4.671
  • 7
    Mayr E, Laule A, Suger G, Ruter A, Claes L. Radiographic results of callus distraction aided by pulsed low-intensity ultrasound. J Orthop Trauma. 2001;15(6):407-14. https://doi.org/10.1097/00005131-200108000-00005
    » https://doi.org/10.1097/00005131-200108000-00005
  • 8
    Pilla AA, Mont MA, Nasser PR, Khan SA, Figueiredo M, Kaufman JJ, Siffert RS. Non-invasive low-intensity pulsed ultrasound accelerates bone healing in the rabbit. J Orthop Trauma. 1990;4(3):246-53. https://doi.org/10.1097/00005131-199004030-00002
    » https://doi.org/10.1097/00005131-199004030-00002
  • 9
    Wu L, Lin L, Qin YX. Enhancement of cell ingrowth, proliferation, and early differentiation in a three-dimensional silicon carbide scaffold using low-intensity pulsed ultrasound. Tissue Eng Part A. 2014;21(1-2):53-61. https://doi.org/10.1089/ten.TEA.2013.0597
    » https://doi.org/10.1089/ten.TEA.2013.0597
  • 10
    Iwai T, Harada Y, Imura K, Iwabuchi S, Murai J, Hiramatsu K, Myoui A, Yoshikawa H, Tsumaki N. Low-intensity pulsed ultrasound increases bone ingrowth into porous hydroxyapatite ceramic. J Bone Miner Metab. 2007;25(6):392-9. https://doi.org/10.1007/s00774-007-0777-5
    » https://doi.org/10.1007/s00774-007-0777-5
  • 11
    Guo X, Jiang H, Zong X, Du L, Zhao J, Zhang D, Song G, Jin X. The implication of the notch signaling pathway in biphasic calcium phosphate ceramic-induced ectopic bone formation: a preliminary experiment. J Biomed Mater Res A. 2020;108(5):1035-44. https://doi.org/10.1002/jbm.a.36878
    » https://doi.org/10.1002/jbm.a.36878
  • 12
    Manchón A, Alkhraisat M, Rueda-Rodriguez C, Torres J, Prados-Frutos JC, Ewald A, Gbureck U, Cabrejos-Azama J, Rodriguez-Gonzalez A, Lopez-Cabarcos E. Silicon calcium phosphate ceramic as novel biomaterial to simulate the bone regenerative properties of autologous bone. J Biomed Mater Res A. 2015;103(2):479-88. https://doi.org/10.1002/jbm.a.35196
  • 13
    Wang L, Zhang B, Bao C, Habibovic P, Hu J, Zhang X. Ectopic osteoid and bone formation by three calcium-phosphate ceramics in rats, rabbits and dogs. PLoS One. 2014;9(9):e107044. https://doi.org/10.1371/journal.pone.0107044
    » https://doi.org/10.1371/journal.pone.0107044
  • 14
    Jiang X, Savchenko O, Li Y, Qi S, Yang T, Zhang W, Chen J. A review of low-intensity pulsed ultrasound for therapeutic applications. IEEE Trans Biomed Eng. 2019;66(10):2704-18. https://doi.org/10.1109/TBME.2018.2889669
    » https://doi.org/10.1109/TBME.2018.2889669
  • 15
    Leung KS, Lee WS, Tsui HF, Liu PP, Cheung W. Complex tibial fracture outcomes following treatment with low-intensity pulsed ultrasound. Ultrasound Med Biol. 2004;30(3):389-95. https://doi.org/10.1016/j.ultrasmedbio.2003.11.008
    » https://doi.org/10.1016/j.ultrasmedbio.2003.11.008
  • 16
    Yang SW, Kuo CL, Chang SJ, Lin YT, Manousakas I, Kuo SM. Does low-intensity pulsed ultrasound treatment repair articular cartilage injury? A rabbit model study. BMC Musculoskelet Disord. 2014;15:36. https://doi.org/10.1186/1471-2474-15-36
    » https://doi.org/10.1186/1471-2474-15-36
  • 17
    Zhou HY, Li Q, Wang JX, Xie YJ, Wang SQ, Lei L, Gao YQ, Huang MM, Hu Y, Xu FY, Zhang C. Low-intensity pulsed ultrasound repair in mandibular condylar cartilage injury rabbit model. Arch Oral Biol. 2019;104:60-6. https://doi.org/10.1016/j.archoralbio.2019.05.018
    » https://doi.org/10.1016/j.archoralbio.2019.05.018
  • 18
    Zhao L, Feng Y, Hu H, Shi A, Zhang L, Wan M. Low-intensity pulsed ultrasound enhances nerve growth factor-induced neurite outgrowth through mechanotransduction-mediated ERK1/2-CREB-Trx-1 signaling. Ultrasound Med Biol. 2016;42(12):2914-25. https://doi.org/10.1016/j.ultrasmedbio.2016.07.017
    » https://doi.org/10.1016/j.ultrasmedbio.2016.07.017
  • 19
    Cui W, Li H, Guan R, Li M, Yang B, Xu Z, Lin M, Tian L, Zhang X, Li B, Liu W, Dong Z, Wang Z, Zheng T, Zhang W, Lin G, Guo Y, Xin Z. Efficacy and safety of novel low-intensity pulsed ultrasound (LIPUS) in treating mild to moderate erectile dysfunction: a multicenter, randomized, double-blind, sham-controlled clinical study. Transl Androl Urol. 2019;8(4):307-19. https://doi.org/10.21037/tau.2019.07.03
    » https://doi.org/10.21037/tau.2019.07.03
  • 20
    Zura R, Della Rocca GJ, Mehta S, Harrison A, Brodie C, Jones J, Steen RG. Treatment of chronic (>1 year) fracture nonunion: heal rate in a cohort of 767 patients treated with low-intensity pulsed ultrasound (LIPUS). Injury. 2015;46(10):2036-41. https://doi.org/10.1016/j.injury.2015.05.042
    » https://doi.org/10.1016/j.injury.2015.05.042
  • 21
    Lou S, Lv H, Li Z, Tang P, Wang Y. Effect of low-intensity pulsed ultrasound on distraction osteogenesis: a systematic review and meta-analysis of randomized controlled trials. J Orthop Surg Res. 2018;13(1):205. https://doi.org/10.1186/s13018-018-0907-x
    » https://doi.org/10.1186/s13018-018-0907-x
  • 22
    Zura R, Mehta S, Della Rocca GJ, Jones J, Steen RG. A cohort study of 4,190 patients treated with low-intensity pulsed ultrasound (LIPUS): findings in the elderly versus all patients. BMC Musculoskelet Disord. 2015;16:45. https://doi.org/10.1186/s12891-015-0498-1
    » https://doi.org/10.1186/s12891-015-0498-1
  • 23
    Hannemann PF, Mommers EH, Schots JP, Schots JP, Brink PR, Poeze M. The effects of low-intensity pulsed ultrasound and pulsed electromagnetic fields bone growth stimulation in acute fractures: a systematic review and meta-analysis of randomized controlled trials. Arch Orthop Trauma Surg. 2014;134(8):1093-106. https://doi.org/10.1007/s00402-014-2014-8
    » https://doi.org/10.1007/s00402-014-2014-8
  • 24
    Leighton R, Watson JT, Giannoudis P, Papakostidis C, Harrison A, Steen RG. Healing of fracture nonunions treated with low-intensity pulsed ultrasound (LIPUS): a systematic review and meta-analysis. Injury. 2017;48(7):1339-47. https://doi.org/10.1016/j.injury.2017.05.016
    » https://doi.org/10.1016/j.injury.2017.05.016
  • 25
    Busse JW, Bhandari M, Einhorn TA, Schemitsch E, Heckman JD, 3rd RT, Leung KS, Heels-Ansdell D, Makosso-Kallyth S, Rocca GJD, Jones CB, Guyatt GH. Re-evaluation of low intensity pulsed ultrasound in treatment of tibial fractures (TRUST): randomized clinical trial. BMJ. 2016;355:i5351. https://doi.org/10.1136/bmj.i5351
    » https://doi.org/10.1136/bmj.i5351
  • 26
    Fan B, Guo Z, Li X, Li S, Gao P, Xiao X, Wu J, Shen C, Jiao Y, Hou W. Electroactive barium titanate coated titanium scaffold improves osteogenesis and osseointegration with low-intensity pulsed ultrasound for large segmental bone defects. Bioact Mater. 2020;5(4):1087-101. https://doi.org/10.1016/j.bioactmat.2020.07.001
    » https://doi.org/10.1016/j.bioactmat.2020.07.001
  • 27
    Xiao D, Zhang J, Zhang C, Barbieri D, Yuan H, Moroni L, Feng G. The role of calcium phosphate surface structure in osteogenesis and the mechanisms involved. Acta Biomater. 2020;106:22-33. https://doi.org/10.1016/j.actbio.2019.12.034
    » https://doi.org/10.1016/j.actbio.2019.12.034
  • 28
    Ebrahimi M, Botelho M. Biphasic calcium phosphates (BCP) of hydroxyapatite (HA) and tricalcium phosphate (TCP) as bone substitutes: importance of physicochemical characterizations in biomaterials studies. Data Brief. 2017;10:93-7. https://doi.org/10.1016/j.dib.2016.11.080
    » https://doi.org/10.1016/j.dib.2016.11.080
  • 29
    Tassinary JAF, Lunardelli A, Basso BS, Dias HB, Catarina AV, Stulp S, Haute GV, Martha BA, Melo DAS, Nunes FB, Donardio MVF, Oliveira JR. Low-intensity pulsed ultrasound (LIPUS) stimulates mineralization of MC3T3-E1 cells through calcium and phosphate uptake. Ultrasonics. 2018;84:290-5. https://doi.org/10.1016/j.ultras.2017.11.011
    » https://doi.org/10.1016/j.ultras.2017.11.011
  • 30
    Winkler T, Sass FA, Duda GN, Schmidt-Beek K. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: the unsolved challenge. Bone Joint Res. 2018;7(3):232-43. https://doi.org/10.1302/2046-3758.73.BJR-2017-0270.R1
    » https://doi.org/10.1302/2046-3758.73.BJR-2017-0270.R1
  • 31
    Ishihara Y, Ueki K, Sotobori M, Marukawa K, Moroi A. Bone regeneration by statin and low-intensity pulsed ultrasound (LIPUS) in rabbit nasal bone. J Craniomaxillofac Surg. 2014;42(3):185-93. https://doi.org/10.1016/j.jcms.2013.05.002
    » https://doi.org/10.1016/j.jcms.2013.05.002

Publication Dates

  • Publication in this collection
    20 Feb 2023
  • Date of issue
    2023

History

  • Received
    02 July 2022
  • Reviewed
    06 Sept 2022
  • Accepted
    01 Oct 2022
Sociedade Brasileira para o Desenvolvimento da Pesquisa em Cirurgia https://actacirbras.com.br/ - São Paulo - SP - Brazil
E-mail: actacirbras@gmail.com