A single dose of P188 prevents cell death in meniscal explants following impact injury

Abstract

Objective

To determine the efficacy of single and multiple administrations of Poloxamer 188 (P188) in saving meniscal cells following an injurious impact.

Methods

Meniscal explants were harvested from both the lateral and medial menisci of Flemish Giant rabbits. After a 24-h incubation period, explants were subjected to 50% impact strain to simulate traumatic joint injury, and the explants were then placed in media with or without supplemented P188. Temporal administrations of P188 over a 14-day period were given based on one of 6 different treatments regimes. Over the 14-day period, explants were cyclically loaded to 10% strain at 1 Hz for 1 h per day, five days a week. Cell viability was assessed on day 14, with the remainder of the tissue being fixed to determine cell apoptosis levels and proteoglycan changes via histology.

Results

The injurious impact proved to produce significant levels of cell death in meniscal explants. The ability of P188 to prevent cell death was not affected by the number of P188 doses (single versus multiple). P188 treatment proved to maintain cell viability levels comparable to those from unimpacted explants. There were no significant changes in cell apoptosis or proteoglycan coverage in the tissues over a 14-day period for any group, all treatment groups were statistically similar to the unimpacted explants.

Conclusion

A single dose of P188 following impact is all that is necessary to inhibit cell death in the meniscus following a traumatic impact. Thus, orthopaedic surgeons may choose to administer P188 in addition to treating any other acute damage due to a traumatic load to the knee, such as anterior cruciate ligament rupture, although more in depth in vivo studies are necessary.

Introduction

Menisci are fibro-cartilaginous structures that aid in joint stability, load distribution and act to protect the underlying articular cartilage from large pressures in the knee joint. Traumatic injuries to the knee joint often lead to damaged menisci (Nielsen and Yde, 1991; Majewski et al., 2006; Hede et al., 1990). It has been long established that injuries to soft tissues of the knee can induce post-traumatic osteoarthritis (PTOA) (Englund and Lohmander, 2004; Englund et al., 2009; Mills et al., 2008; Katz and Martin, 2009), although the exact mechanisms to the onset of the disease remains unknown. Previous studies have shown that damage to the menisci can lead to early degeneration of the underlying cartilage (Crema et al., 2010; Hunter et al., 2006; McCann et al., 2009), and a 15-fold increase in the likelihood of a patient developing end stage osteoarthritis and undergoing total knee replacement (Khan et al., 2018), whereby highlighting the importance of these structures within the knee joint. Current clinical treatment for meniscal injuries focuses on macroscopic acute tissue damage by removing the damaged portions, but it neglects any occult microscopic damage that may prove crucial in the development of PTOA. Pharmaceutical intervention that mitigates the progression of occult damage on the cellular level following a traumatic impact may be able to help mitigate further damage to the menisci and avert the progression of PTOA.

Poloxamer 188 (P188) is an amphipathic, non-ionic surfactant that has been shown to prevent cell death in neurons (Lee et al., 1999) and cartilage (Phillips and Haut, 2004; Baars et al., 2006; Natoli and Athanasiou, 2008; Bajaj et al., 2010; Pascual Garrido et al., 2009; Isaac et al., 2010; Rundell et al., 2005). P188 is a tri-block polymer consisting of hydrophilic ends and a hydrophobic body, thus allowing itself to insert within the lipid bilayer of cell membranes to restore their integrity after physical stress. Previous in-vitro studies have demonstrated the ability of a single 8 mg/mL dose of P188, administered immediately following impact, in preserving cell viability in articular cartilage (Phillips and Haut, 2004; Baars et al., 2006; Natoli and Athanasiou, 2008; Bajaj et al., 2010; Pascual Garrido et al., 2009). Similarly, two animal impact models report that P188 significantly reduces the amount of cell death in articular cartilage, when compared to untreated groups (Isaac et al., 2010; Rundell et al., 2005). Multiple studies suggest that timing additional P188 administrations to coincide with cell apoptosis would potentially improve the efficacy of the surfactant (Phillips and Haut, 2004; Baars et al., 2006; Natoli and Athanasiou, 2008; Isaac et al., 2010; Rundell et al., 2005). To the best of the authors’ knowledge, only one study has evaluated the efficacy of the surfactant on meniscal tissue. In a closed-joint animal impact model, Coatney et al. report that at six weeks post injection, a single intraarticular injection of P188 significantly helped to preserve the proteoglycan content of the meniscus when compared to menisci from an untreated impact group (Coatney et al., 2015). Although the study did not report data on cell viability, the results show that P188 is a viable option in preserving the integrity of other soft tissues in the knee joint. It is not clear if the P188 injection directly affected meniscal changes or if it was indirectly through effects on other tissues in the knee joint. Thus, to better understand the actions of P188 on meniscal tissue directly, it is necessary to run isolated in vitro studies with isolated meniscal tissue to determine the direct effects of P188 on meniscus in the absence of other joint tissues. The current literature suggests that multiple administrations of P188 post-impact, that coincide with cell necrosis and apoptosis time points, may be necessary to maintain meniscal cell membrane integrity following the acute injury, and potentially slow or halt the progression of PTOA.

The objective of the current study therefore, is to characterize the effects of single and multiple temporal administrations of P188 to meniscal explants after being subjected to a traumatic impact. The administration time points of immediate (time 0) (Ewers et al., 2001), 4 days (Costouros et al., 2004) and 7 days (Pascual Garrido et al., 2009) post-impact were chosen to coincide with previously reported high levels of cell necrosis and apoptosis. It was hypothesized that P188 would reduce cell death, thereby maintaining the biochemical integrity of the tissue similar to that of an unimpacted control group. This would be the first in-vitro study to assess the efficacy of P188 on cell viability and tissue biochemistry in meniscal tissue.