“Am I going to be able to walk again without help?”

As a specialist in adult reconstruction, Amanda D. Marshall, MD, hears this question nearly every day.

“Total knee and total hip arthroplasty are some of the most life-changing procedures in all of medicine,” said Dr. Marshall, assistant professor in the department of orthopaedics at the University of Texas at San Antonio and three-time Orthopaedic Research and Education Foundation (OREF) grant recipient. “Patients are ecstatic when their quality of life is so improved after joint replacement, and the fulfillment I get from that is unparalleled.”

“I’ve always been interested in the impact of wear debris on the surrounding bone and soft tissue,” said Dr. Marshall. “A large percentage of my surgical procedures also deals with osteolytic lesions, so I’d like to find ways to minimize the detrimental effects of wear debris down the road—ideally to the point where patients will never need revision surgery.”

Two OREF grants (a 2007 Zimmer Orthopaedic Career Development Award and a 2011 ORS/OREF Travel Award in Orthopaedic Research Translation) gave Dr. Marshall opportunities to work with top adult reconstruction clinicians and scientists who share her interest in investigating particle-induced osteolysis, especially the effect of ultra-high molecular weight polyethylene (UHMWPE) particles.

Encouraged by results of research she conducted to illustrate a direct effect of the wear debris on osteoblasts, Dr. Marshall applied for an additional OREF grant to support further study.

“My preliminary data were very exciting. They showed that these polyethylene particles definitely affect certain osteoblast cell types,” she said. With help from a 2011 AAHKS/OREF Research Grant in Adult Reconstruction, Dr. Marshall is now investigating that phenomenon with the bone-forming cell’s precursor—the stem cell. The long-term goal is to develop several alternatives to revision surgeries associated with bone loss and subsequent aseptic loosening.

Does size matter?
Little is known of the effects of particulate debris on mesenchymal stem cells (MSCs) and subsequent osteoblastic differentiation. Dr. Marshall’s goal is to determine the impact of both UHMWPE particles of different sizes and overall particle load on the bone-forming potential of human MSCs. Using an isolation method that she and her institutional collaborators developed, Dr. Marshall will examine nanometer-, submicron-, and micron-sized UHMWPE particles. The influence of these particles on the natural function of MSCs will then be quantified.

“Our hope is that we will be able to determine whether a size-dependent effect exists, and then possibly design materials that produce less of a specific size of particle,” Dr. Marshall explained. “We may also find a way to modify the surrounding tissues to make them less susceptible to damage by these particular particle sizes and loads.”

Step by step
To obtain UHMWPE particles, commercially available GUR 1050 resin will be suspended in pH 5.5 water containing Pluronic and then vortexed for 15 minutes, sonicated for 120 minutes, and stored at 4°C for 7 days. The particles will then be fractionated into the three size subpopulations by vacuum filtration. Particle analysis will be performed via electron microscopy on all subpopulations to define size and shape according to ASTM standards.

Human bone marrow mononuclear cells (which contain MSCs) will be obtained from 20- to 30-year-old donors. The cells will be cultured on tissue culture plastic until confluent. They will be expanded for 7 days under the following conditions:

• negative control
• nanoparticles
• submicron particles
• micron particles
• positive control (unfractionated particles)

Cells will then be examined to determine the effects of UHMWPE particle size and dose on MSC replication, differentiation into mature osteoblasts, and bone-forming capacity in an in vivo mouse model.

“We hypothesize that the particles will have both a size- and dose-dependent effect on human MSCs, with nanometer-size particles having the most deleterious effect on the cells,” said Dr. Marshall. “If we can determine the impact of the particles on the actual cells, and the process by which they affect these cells, perhaps we can target a potential step in the process that will either reverse that process of bone loss, or somehow stimulate the osteoblast progenitor to turn those stem cells back into bone-forming cells.”

Fewer revisions
With an understanding of the exact effects of UHMWPE particles on MSCs, treatment strategies can be developed that target specific molecular mechanisms that contribute to osteolysis, possibly a downregulation of osteolytic signaling, or an upregulation of osteoblastic response. Alternatively, treatments might emerge that will modify surrounding bone and soft tissue to make them less susceptible to damaging particles.

In addition, knowing the size-dependent effects of particulate debris can guide device manufacturers in refining their choice of materials and manufacturing processes to minimize or eliminate the release of the most harmful particle sizes. Another potential application of the study’s findings is development of a preoperative risk assessment instrument that will predict a joint arthroplasty candidate’s response to the anticipated particle load by advanced screening tests.

“Our goal is to lay some solid groundwork on this particular material’s impact on stem cells,” said Dr. Marshall. “Perhaps an improved understanding of the interaction of particles and stem cells—or the biological mechanism of osteolysis—will bring us a little closer to solving the problem of aseptic loosening so we won’t have to perform as many revisions. The ultimate goal of our research is to implement new knowledge that results in increased longevity of prostheses, lower rates of osteolysis development, and ultimately improved patient outcomes.

“One great thing about the OREF grant is that it has allowed me to work across several departments of my institution—dentistry, pathology, the Southwest Research Institute—which has been instrumental in catapulting our lab’s efficiency and reputation,” continued Dr. Marshall. “Our lab and our department are so very thankful to OREF and all of its contributors for this opportunity to enhance our understanding of implant wear. Truly, OREF keeps alive the dream of conducting multidisciplinary clinically relevant basic science research.”

Mark Crawford is a contributing writer for OREF and can be reached at communications@oref.org