This raised the questions, “Is there an age-related decline in the ability of chondrocytes to maintain and restore articular cartilage? Could this change contribute to the age-related increase in the incidence of osteoarthritis?”


Published 6/1/2007
Joseph A. Buckwalter, MD

Basic research and the future of orthopaedics

Editor’s note: Joseph A. Buckwalter, MD, is the recipient of the 2007 Orthopaedic Research Society (ORS)/American Orthopaedic Association (AOA) Alfred R. Shands Jr., MD, Award and Lecture. These excerpts are from his presentation to the ORS 53rd Annual Meeting, Feb. 11-14, 2007, in San Diego, Calif.

When I started studying articular cartilage, it was often referred to as a relatively inactive structural material with a few indolent cells embedded within an amorphous ground substance.

A technique developed by Lawrence C. Rosenberg, MD, enabled researchers to view proteoglycan structure in multiple skeletal tissues. We found that the amorphous appearance of the cartilage ground substance hid a variety of complex, elaborate molecules. We discovered striking differences in proteoglycans, in particular between proteoglycan aggregates—such as the remarkably large elaborate proteoglycans found in intervertebral disks, the tightly packed aggregates found in fetal epiphyseal tissue, and the smaller aggregates found in mature articular cartilage.

Intrigued by these structural differences, we looked at the proteoglycan aggregates of the growth plate. In the epiphyseal and upper reserve zones, the proteoglycan aggregates were very large and held together with densely packed chondroitin sulfate chains. Building on the work of David S. Howell, MD, and Julio Pita, PhD, who showed that the protein polysaccharoid molecules from the upper regions of the growth plate were effective inhibitors of mineralization, we found that the large proteoglycans were lost in the lower regions of the hypertrophic zone in the zone of provisional calcification.

Our observations fit well with the suggestion by Michael G. Ehrlich, MD, that release of degradative enzymes by chondrocytes helped prepare the extracellular matrix for mineralization by removing the inhibitors of mineralization.

Age-related changes in cartilage
In studying articular cartilage from animals and humans of different ages, we found that the articular cartilage proteoglycan aggregates decreased in size, the aggrecans became smaller and more variable, and the chondroitin sulfate chain length decreased as the subjects aged. Further investigations verified that the alterations in aggrecan size and structure and the increasing variability were due to changes in chondrocyte synthetic patterns.

James A. Martin, PhD, and I have been pursuing these questions for nearly a decade. In both humans and animals, chondrocyte synthetic activity declines with age. In addition, the chondrocyte response to anabolic factors declines. When newborn chondrocytes in culture are exposed to the anabolic cytokine IGF-I, they essentially double their synthetic activity. As they age, this positive response decays. Similar changes occur in human chondrocytes.

At last year’s ORS meeting, we reported on our findings that aggrecan gene expression in rat mesenchymal stem cells declined more than 75 percent as cells aged from one week to one year old. This year, Michael Huang, MD, reports that the proteoglycan content of chondral repair tissue in osteochondral defects declines more than 75 percent between 1 month and 12 months of age in a rat model.

Is cell senescence the reason?
These observations raise the question, “Why does chondrocyte function decline with age?”

One possibility is the phenomenon of cell senescence, which was first identified in the late 1800s and described as a loss of power of growth with each successive generation of cells. In the 1960s, Leonard Hayflick, PhD, found that differentiated cells decreased their rate of proliferation in culture after a certain number of population doublings. Eventually, they enter a nonproliferative state from which they never recover, a phenomenon called the Hayflick Limit. Since then, others have found that as cells approach replicative senescence, they lose their differentiated phenotype.

One possible explanation of cell senescence is DNA damage marked by telomere erosion. Telomeres are the DNA sequences that cap the ends of chromosomes and make it possible for DNA telomerase to replicate DNA.

With each cell division, part of the telomere is lost. After more than 80 percent of the telomere sequence is degraded, some cells become senescent. Others have the enzyme telomerase, which rebuilds telomeres and may have a role in DNA repair in general. Telomerase is absent from most cells, but is present in many tumors and in mortal cells.

The role of telomeres in cell tissue and organism function is far from clear, but there are several interesting observations. For example, a study published in the Lancet (2003) found that people with short telomeres had twice the mortality rate from all causes as people with long telomeres.

Human chondrocyte telomere length decreases with age, which raises some challenging questions. For example, because chondrocytes rarely divide after reaching skeletal maturity, does telomere erosion or age-related DNA damage occur by mechanisms other than cell division?

The role of loading in chondrocyte function
Loss of chondrocyte function with age may contribute to the increased risk of osteoarthritis with age, but does not explain how osteoarthritis develops in young adults or in people with joint dysplasia or joint trauma. In these individuals, excessive mechanical loading of the articular surface may cause a loss of chondrocyte function.

In vitro studies show that chondrocytes exposed to high sheer stress have an increased rate of both apoptosis and cell death. High sheer stress stimulates release of oxygen free radicals. Most cells express oxygen free radicals under high-stress conditions, but few do so under low-stress conditions.

Oxygen also causes chondrocyte DNA damage. According to data using a flare assay, 21 percent oxygen causes roughly 2½ times as much DNA damage to chondrocytes as 5 percent oxygen. If mechanically induced chondrocyte death is partly due to oxidative damage, it should be possible to block this effect by blocking oxygen free radicals.

In one experiment, we exposed viable chondrocytes to no load, to mild mechanical stress, and to intense compressive stress. Under no load, most chondrocytes remained viable. Under intense compressive stress, there was a striking increase in the number of dead cells, but this effect was substantially decreased when the tissue was treated with antioxidants, in this case vitamin E. This strongly suggests that at least one mechanism of chondrocyte cell death in these in vitro experiments is oxidative damage.

These observations—sheer stress can cause in vitro chondrocyte death and release of oxygen free radicals, and chondrocytes are susceptible to oxidative damage—suggest that preventing deleterious mechanical stress could reduce the risk of osteoarthritis due to excessive mechanical stress. We will need to know more before we can implement changes in patients with posttraumatic or other forms of osteoarthritis. There may be biologic interventions—such as antioxidants—or other strategies that prevent propagation of mechanical damage.

Immortal chondrocytes?
Although normal human articular cartilage chondrocytes become senescent at about 25 population doublings when they are raised in 21 percent oxygen, if they are transfected with the telomerase gene and oxidative damage is prevented, the chondrocytes become immortal. This presents a challenging clinical problem in patients with chondrosarcomas.

In culture, high-grade human chondrosarcomas express a very high level of the enzyme telomerase. Telomerase staining of chondrosarcoma tissue sections correlates to some degree with classic histologic grading of chondrosarcomas. The highest grade chondrosarcomas, presumably the most malignant, have the highest percentage of cells staining positive for telomerase.

In a small retrospective study, patients who had chondrosarcomas in which more than 75 percent of the cells stained positive for telomerase had a very poor prognosis. Patients who had tumors with low levels of telomerase had a much greater probability of surviving more than five years and perhaps for many decades.

Could blocking telomerase activity in high-grade chondrosarcomas prevent growth? According to papers presented both last year and this year, exposing high-grade chondrosarcomas with high telomerase activity to selective telomerase inhibitors can reduce tumor activity to a level comparable to low-grade chondrosarcomas—perhaps even close to that of normal cartilage.

Will these ideas concerning osteoarthritis and chondrosarcomas lead to improved understanding of these diseases and eventually better treatments? It is difficult to say at this point, but clearly, new ideas from basic research will lead to breakthroughs.

Since I attended my first ORS meeting 31 years ago, research presented at this meeting has transformed our understanding of both cartilage and all musculoskeletal tissues, and has become the driving force in developing new treatments. It is exciting to see the many talented investigators attending this meeting and to know that the future of orthopaedic research will be greater than the past, leading to breakthroughs in the treatment of patients with musculoskeletal diseases and injuries.