Ex-vivo Study of Detergent Based Intraosseous Bone Wash for Treatment of Ischemic Osteonecrosis
Grant Recipient: Graham Andre, BS
Co-Investigators: Chi Ma, PhD; Mo Singhal, BS; Vishal Gokani, BS; Harry K.W. Kim, MD
- Texas Scottish Rite Hospital
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Osteonecrosis (ON) is the death of bone tissue due to a loss of blood supply, which most often affects the hips, shoulders, and knees. ON leads to an abundance of necrotic debris (e.g. hematopoietic cells and adipocytes) left in bone marrow space due to lysis of cells, which forms a necrotic microenvironment idling bone regeneration. We have developed an interosseous saline bone wash to remove these cellular debris and lipids, but the technique has failed to completely remove the necrotic lipids from the epiphysis. Poloxamer 407 (P407) is a triblock copolymer consisting of a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol. P407 is completely removed by the kidney, requires no metabolism, and has no cellular toxicity. P407 can be used as a non-ionic detergent because of its surfactant properties, which can improve the solubility of lipid ingredients in an aqueous solution. In this study, we aimed to determine the efficiency of the non-toxic detergent P407 as a bone wash medium in the removal of necrotic lipids. Our HYPOTHESIS was that 10% poloxamer 407 would extract more lipids, cellular debris, and increase washing efficiency from the trabecular meshwork space when compared to saline controls.
Humeral heads from harvested piglets (37-42 lbs) underwent three freeze-thaw cycles, causing cells to lyse and release damage associated molecular particles, cellular debris, and necrotic lipids into the marrow space thus mimicking the necrotic process of ON. Two self-drilling fenestrated needles were placed using a drill and guide for 12mm inter-needle distance. This distance was chosen based on previous studies to determine the most effective washing based on inter-needle distance. Correct placement of the needles distal to the epiphyseal plate was confirmed using X-ray. Each head was washed with either P407 or saline. 30ml syringes were attached to the ends of the needles, one needle was used as an inflow needle and the other for extracting the wash medium for analysis. The needles alternated inflow and outflow to allow for thorough washing. Wash fractions of the extracted medium were frozen and underwent frozen dried lyophilization. At first only a small fraction was used for lyophilization (1ml/wash), however after subtracting out the saline and poloxamer weight we realized we needed a larger fraction (5ml/wash) so that small sampling errors would not effect the accuracy of the experimental design. Other wash fractions were used to measure extracted lipids using Sigma-Aldrich Lipid Triglyceride Quantification. Preliminary testing showed that the first four washes showed significant step wise decrease in lipid quantification as expected, but that the following washes (5-15) did not have a significant difference between each subsequent wash. Since the trend of decreasing concentration of each wash was not important to the experiment, we modified the design to combine the washes fractions from each head sample to obtain a total amount of lipids removed from each head, this was done in both saline vs poloxamer detergent samples. The washed heads were fixed in formalin and then sliced into six, 5mm pieces, 3 anterior and 3 posteriors using a 3D printed guide and band saw. Gross coronal section scans of the humeral heads were analyzed using OSTEOIMAGER (BIOQUANT Image Analysis, TN) to quantify washing efficiency of poloxamer vs saline using average pixel light intensity (grey value) and percent washed within a region of interest. Percent cleaned grey value was determined using histology to confirm the trabecular meshwork was void of all necrotic debris .
In all quantitative measures, poloxamer 407 was superior to saline controls as a wash medium. P407 wash medium extracted significantly more lipids (p=0.006) from experimental heads (N=5) than heads washed with saline controls (N=5). Lyophilization data yielded a significant increase in cellular debris removed (p=0.02) from the heads in the poloxamer samples (N=4) when compared to the saline control group (N=4). Wash quantification of the head slices showed a significant increase in both grey value (overall cleanliness of the slices) (p=0.03) and percent cleaned (specific grey value threshold met)(p=0.03) in the experimental poloxamer group (N=6) compared to saline group (N=6). Our hypothesis was not disproven as the quantitative data suggest that poloxamer is a superior wash medium in extracting insoluble cellular debris and necrotic lipids from the interosseous marrow space.
ON promotes a pro-inflammatory environment by release of cytokines, necrotic lipids and proteins that hinders pro-osteogenic growth. The end goal of this research is to provide a safe, effective, and minimally invasive procedure to help reverse the necrotic environment that occurs in ON by removing these pro-inflammatory cellular debris, lipids, and DAMPs. This research laid the foundation using an ex vivo model to support the use of poloxamer as a wash medium. Future studies will use an in vivo approach on pigs to show the efficacy and safety of this approach leading to future clinical applications in treatment of ON, specifically Legg Perthes disease.
The abstract from this research was submitted to the 59th Annual Medical Student Research Forum at UTSouthwestern Medical School, it was selected to be one of the four featured oral presentations for the symposium, the presentation took place on Feb 2nd, 2021. The abstract was also submitted to and selected for a podium presentation at the 2022 Orthopedic Research Symposium Annual meeting on Feb 4th – 8th in Tampa, Florida.