(176d) Low-Intensity Continuous Ultrasound Promotes Healing of Damaged Cartilage in a Pro-Inflammatory Environment

Low-Intensity Continuous Ultrasound
Promotes Healing of Damaged Cartilage in a Pro-Inflammatory Environment

Neety Sahu¹, Hendrik Viljoen¹
and Anuradha Subramanian^1*

¹University of
Nebraska-Lincoln, Lincoln, NE 68588-0643

*asubramanian2@unl.edu

Repair of damaged cartilage
remains a biomedical burden and a research challenge. Cartilage repair
techniques including tissue engineered approaches have failed to generate
long-lasting hyaline cartilage that meets functional demands. Inflammation at
the damage site or the invasive operating method itself may cause additional
pro-inflammatory cytokine responses. Hence cartilage repair takes place in a
joint environment, with elevated levels of proinflammatory cytokines, that is
known to impede repair and lead to cartilage destruction. Thus, immunomodulation
approaches have been devised to achieve cartilage repair in proinflammatory
environment.  Pulsed Low-Intensity-Ultrasound (pLIUS) has been reported to
possess chondro-regenerative and chondroprotective effects in studies limited
to in-vitro and ex-vivo studies of intact cartilage and
chondrocytes.  

In
a departure from all previous studies that employ pLIUS, our approach employs
continuous LIUS (cLIUS) as it couples more energy when compared to pulsed LIUS
and it does so at the cell resonant frequency of 5 MHz where cLIUS induced
bioeffects are maximized^{1, 2}. Thus a treatment modality based on cLIUS
at the resonant frequency of 5MHz is proposed for promoting cartilage repair in
a pro-inflammatory environment using an established in-vitro model of cartilage
damage. Our study is predicated on the hypothesis that cLIUS enhances
chondrocyte phenotype and cellular migration and inhibits the expression of
catabolic markers, notably in a pro-inflammatory environment. We have used osteochondral
explants with cylindrical incisions to simulate cartilage lesions. The incised explants
were maintained under LIUS stimulation (5 MHz, (14 kPa, 20 minutes, 4 times per
day) in the cLIUS-assisted bioreactor³ developed at the University
of Nebraska-Lincoln both in the presence and absence of pro-inflammatory
cytokines, IL6 and TNFα for a period of 28-days. Non-stimulated explants
served as controls. Explant specimens were evaluated histologically and
immunohistochemically⁴
over a period of 28 days. Gene expression of various chondrocytic markers and
markers for catabolic damage were assessed by qRT-PCR⁴.

Histological assessment of the explants displayed closed gaps and
maintenance of tissue continuity in LIUS treated explants (panel IV) whereas
visible gaps remained in non LIUS-treated controls (panel I). Cartilage-to-cartilage
bonding was also observed in osteochondral explants exposed to IL-6 or TNF-a and treated with cLIUS (panels V and VI). Fissure
or a cleft was observed in osteochondral explants exposed to IL-6 or TNF-a and not exposed to cLIUS (panels II and
III). These cytokines are known to promote loss of glycosaminoglycan (GAG) from
the matrix and this was visible in explants treated with cytokines and not
treated with cLIUS (panels II and III). GAG loss to the medium was also
measured and a similar trend was noted (Fig. 1B). When compared to controls,
cLIUS was observed to enhance migration of cells to the leading edge of a
defect in both 2D and 3D in-vitro assessments both in the presence and absence
of IL6 and TNfa (data not included). cLIUS was also noted to enhance the
expression of chondrocytic markers COL2A1 both in the presence and absence of
cytokines, when compared to control (data not included).

As expected, exposure of osteochondral explants to pro-inflammatory
cytokines resulted in over-expression of MMP13 and ADAMTS4 and ADAMTS5, known
matrix metalloproteinases and catabolic agents (Fig. 2A-B). Interestingly,
cLIUS was observed to suppress the expression of MMP13 and ADAMTS4 and ADAMTS5
in osteochondral specimens exposed to IL6 and TNFa
(Fig. 2A/B); while increasing
the expression of TIMP1, an inhibitor of metalloproteinases (Fig. 2C).

In conclusion, our combined results demonstrate that cLIUS when
applied at the resonant frequency can promote cartilage repair in a
pro-inflammatory environment by inhibiting the expression of catabolic factors
and by promoting the chondrocytic phenotype.

References:

1.         Miller AD, Subramanain, A. and Viljoen, H. J. A nonlinear model of cell
interaction with an acoustic field. Journal of Biomechanics 2017 14
March 2017. DOI: http://dx.doi.org/10.1016/j.jbiomech.2017.03.007.

2.         Miller
AD, Chama A, Louw TM, et al. Frequency sensitive mechanism in low intensity
ultrasound enhanced bioeffects. PloS one 2017; 12. DOI: ARTN e0181717.
10.1371/journal.pone.0181717.

3.         Subramanian
A, Turner JA, Budhiraja G, et al. Ultrasonic bioreactor as a platform for
studying cellular response. Tissue engineering Part C, Methods 2013; 19:
244-255. PMCID 3557434. DOI: 10.1089/ten.TEC.2012.0199.

4.         Guha Thakurta S, Sahu, N., Budhiraja,
G., Miller, A. D., Akert, L., Viljoen, H. J. and Subramanian. A. Long-term
culture of human mesenchymal stem cell-seeded constructs under ultrasound
stimulation: evaluation ofchondrogenesis. Biomed Phys Eng Express 2 (2016)
055016 2016; 2: http://dx.doi.org/10.1088/2057-1976/1082/1085/055016.