What’s New in Orthopaedic Research

Exciting advances continue to be made in numerous areas of orthopaedic research. One of the most important areas of research continues to be the discovery of biologic solutions to degenerative joint disease. Emerging information about the genetic basis of bone and cartilage pathology is being used to provide insight into the fundamental mechanisms of common conditions such as osteoporosis, degenerative disc disease, and arthritis. Other active areas of investigation include stem cells, growth factors, tissue engineering, biomaterials, and the biomechanical properties of tissues at the nanoscale. Another common theme is improving the understanding of the basic cellular mechanisms that control the response of musculoskeletal tissues to mechanical load. Investigations in these areas have the potential to improve current treatments and to lead to the development of novel therapies and implants. This paper reviews several major areas of active investigation.

Lessons from Transgenic Mice and Human Genetic Diseases

Bone and Skeletal Development

A study of mice deficient in membrane-type 1 matrix metalloproteinase (MT1-MMP) showed that MT1-MMP plays a fundamental role in a novel mechanism of remodeling of unmineralized cartilage anlagen into membranous bone, ligament, and tendon1. While the most common mechanisms of remodeling in the skeleton (endochondral ossification and postnatal bone remodeling) involve replacement of a mineralized matrix by a newer mineralized matrix, in this newly discovered process MT1-MMP directs dissolution of unmineralized cartilage and apoptosis of nonhypertrophic chondrocytes. This process occurs without progression through the sequence of matrix mineralization. MT1-MMP also allows remodeling of cartilage into ligament at insertion sites. MT1-MMP is expressed at ligament insertion sites, implying that this molecule likely plays an important role in soft tissue-bone attachment.

Human genetic diseases can provide a mechanism for identifying genes that are important for bone formation. Genetic studies also may shed light on the sensitivity of bone to mechanical loading. It is known that some individuals respond better to exercise (mechanical loading) than others do. The mechanosensitivity of bone is affected by genetics. A symposium at the recent meeting of the Orthopaedic Research Society reviewed three general methods for identifying genes that contribute to traits such as bone fragility and to the mechanical loading response of bone. The first approach involves identifying individuals who have an altered skeletal response that is linked to mutations (or polymorphisms) in a single gene. Typically, these are rare genetic mutations. A good example was recently reported following an examination of patients with sclerosteosis, which is a rare skeletal disorder that is characterized by high bone mass due to increased osteoblastic activity. Recent studies have shown that sclerosteosis results from loss of the SOST gene product sclerostin2. Sclerostin is exclusively expressed in osteocytes and suppresses bone formation by inhibiting osteoblast differentiation. This secreted protein is transported to the bone surface, where it inhibits osteoblasts. Inactivation of sclerostin may provide a valuable treatment option for patients with osteoporosis.

Another approach for identifying genes that contribute to traits such as bone fragility employs inbred strains of mice that differ in terms of bone mineral density or bone strength. Matings between mice from two inbred strains allow for the segregation of genetic alleles that are important for bone structure or strength. This approach allows for the identification of quantitative trait loci for bone mineral density. Once these loci are identified, each one can be isolated in a mouse strain for future study. Because the long bones of C57BL/6J mice are larger in cross-section and are more responsive to mechanical loading than are the bones from C3H/HeJ mice, C57BL/ 6J mice have been used to provide a means for mapping quantitative trait loci associated with skeletal mechanosensitivity3.

A third approach for identifying genes contributing to bone strength involves the identification of candidate genes. One recently reported candidate gene is the P2X7 receptor, which is an ATP-gated ion channel4. Mice with a null mutation of the P2X7 receptor were shown to have reduced bone mass and reduced periosteal bone formation. These findings suggest that the P2X7 receptor functions in bone mechanotransduction.

Tendon

Transgenic mice are now being used to identify candidate genes that govern tendon structure and composition and that influence adaptation to mechanical loading and healing. Recent studies have shown that the biomechanical and morphological properties of tendon vary significantly with genotype. These differences are not explained by mouse size or tendon size, indicating that unique structural and compositional factors play a role in tendon growth and development. Soslowsky and colleagues examined tendons from decorin and biglycan knock-out mice5,6. They found that patellar tendons lacking decorin had an increased modulus of elasticity and an increased amount of stress relaxation. In all cases, failure occurred at the insertion site rather than in the mid-substance of the tendon during tensile testing, indicating that loss of decorin and biglycan affects the insertion site more than the midsubstance of the tendon. These investigators also reported that the absence of decorin and biglycan affects tendon mechanical properties differently for different tendons in the same animal, indicating that the relationship of composition to function is more complex than a simple relationship of one molecule to tensile properties. These investigators further examined the role of interleukin-4 (IL- 4) and interleukin-6 (IL-6) in tendonhealing. Because IL-6 is a potent proinflammatory molecule, it might be expected that mice lacking IL-6 would have improved tendon-healing due to less inflammation, more fibroblastic activity, and the production of more organized collagen. However, the IL-6 knock-out mice did not demonstrate superior healing, likely because of compensatory mechanisms. These results point out the complex molecular mechanisms of tendon-healing and the need for additional studies.

Gene and Stem-Cell Therapy

Advances in stem-cell therapy have brought clinical validation as well as new scientific questions to the fore. Clinically, bone marrow-derived cells have been used to improve the growth of children with osteogenesis imperfecta7, to induce partial local restoration of dystrophin expression in patients with muscular dystrophy, and to improve circulatory function in individuals with ischemic limbs8. Even in the face of these successes, however, questions about whether and how bone marrow-derived cells may transdifferentiate into nonlymphohematopoietic tissue persist. Evidence from both cell and organ transplantation studies has shown that circulating and noncirculating cells can engraft into solid organs and tissues. Some evidence now suggests that stem cells may home to sites of injury. Fusion of stem cells to resident cells, followed by reductive cell division, may be one mechanism by which stem cells become differentiated8,9.

While cells with the capacity to self-renew and to differentiate into several different phenotypes have now been isolated from many tissues in addition to bone marrow, the question of whether these are, in fact, “stem cells” remains a matter of intense debate. Research by several groups has shown that stem cells isolated in the same manner from different strains of mice or from different species of rodents (mice and rats) are, in fact, quite different in their regenerative potential10 These discrepancies indicate that the criteria for the isolation and initial characterization of stem cells are not yet completely defined, even in the experimental setting. As reports of the isolation of “stem cells” from a variety of tissues (adipose tissue, synovial tissue, and even articular cartilage) continue to grow and potential clinical applications increase, determining characteristics that strictly define “stem cells” (e.g., cell-surface markers and the capacity for clonal growth and/or transdifferentiation) becomes more and more important.

Regardless of whether or not they are truly stem cells, self- renewing cells from a variety of sources have been shown to be useful as platforms for gene therapy. In a recent study, Chamberlain et al. showed that an adeno-associated vector could be used to eliminate the expression of the mutated type-I collagen gene and increase the expression of normal type-I collagen by marrow-derived stem cells from patients with osteogenesis imperfecta11To emphasize that the cells maintained their “stem-ness,” the researchers showed that the cells were able to differentiate into bone and adipose cells, even after gene-targeting. In a study underscoring the interesting potential interaction between stem cells and transgenes, Peng et al. reported that, for bone repair, the most “stem-like” cells may not be the best platform for delivering the bone morphogenetic protein-4 (BMP-4) gene12 In that study, the most regenerative and plastic muscle-derived stem cells underwent differentiation and apoptosis when they were genetically modified to \overexpress BMP-4, limiting their capacity to participate in bone- healing in a mouse calvarial defect model. In contrast, less plastic yet still regenerative cells derived from muscle in a similar manner were more effective as they did not differentiate or undergo apoptosis in response to the transgene.

Developments in gene-transfer technology that promise the controlled, long-term expression of therapeutic genes also support the continued development of stem cells as a platform for gene delivery. Successful longterm gene transfer has been demonstrated in several cell types with use of lentivirus vectors. Other studies have shown that regulatable promoters such as those that will only allow gene expression in bone tissue (osteocalcin promoter), in the presence of inflammation (IL-l/IL-6 promoter/enhancer), or in the presence of an activating drug (tetracycline-sensitive promoter) can ensure that genes that have been transferred for the long term are only expressed under the desired circumstances13,14.

Cartilage Degradation and Repair

An accumulating body of knowledge about the interactions between chondrocytes and the extracellular matrix proteins of cartilage is shedding new light on the mechanisms underlying cartilage degeneration in osteoarthritis and other arthritides. A symposium at the recent meeting of the Orthopaedic Research Society reviewed the work of several investigators who have found that fragments of extracellular matrix proteins have very different and often deleterious effects when compared with their intact counterparts. Homandberg and colleagues have extensively studied the role of fibronectin fragments (Fn-fs) in inducing chondrolysis (chondrocytemediated cartilage degradation) and recently showed that this interaction is important in human cartilage15. Loeser et al.16 elucidated many of the pathways that are involved in these interactions, including the binding of Fn-fs to specific receptors on the chondrocyte cell surface and the ensuing intracellular cascade that finally induces the expression of several genes, including the collagen-degrading enzyme MMP-13. In mediating these chondrocyte-extracellular matrix interactions, [alpha]5 integrin appears to be of particular importance. Integrins are a group of heterodimeric cell-surface receptors that recognize particular extracellular matrix proteins and that also serve as docking points for several intracellular signaling molecules. The [alpha]5 integrin, probably as a heterodimer with [beta]1 integrin, appears to be particularly important for mediating the signal not only for Fn-fs but also for fragments of type-II collagen, which can also induce MMP-13 expression by articular chondrocytes17. Signaling through CD44 and/or other receptors are fragments of hyaluronic acid, which, unlike their intact counterparts, also can induce chondrolysis.

An interesting recent development in the research on cartilage repair has been the focus on the use of subpopulations of chondrocytes, with the goal of recapitulating the morphologic characteristics of cartilage in engineered tissues. Several investigators have previously shown that chondrocytes at the surface of articular cartilage (superficial zone chondrocytes) are phenotypically distinct, expressing, for example, the lubricating proteoglycan surface zone protein, which is not expressed by chondrocytes deeper within the cartilage tissue. Recent studies from two laboratories focused on the construction of tissue-engineered cartilage constructs that recapitulate the stratified morphology of articular cartilage by layering engineered tissue made of superficial zone chondrocytes on top of that consisting of chondrocytes from deeper within cartilage tissue18,19. These studies showed that this strategy can modulate the mechanical properties of the tissue that is generated. Of particular interest, a study by Sharma et al. showed the apparent suppression of deep zone chondrocyte proliferation by the superficial cells, suggesting that paracrine signals between the two subpopulations may be important in maintaining the morphology of cartilage tissue20. In addition, a recent report by Dowthwaite et al. suggested that the use of superficial zone chondrocytes provides a cell layer with different mechanical properties that not only secretes distinct physiologic factors but also may be enriched in stem-like cells21.

The Role of Pharmacological Agents in Fracture-Healing and Implant Fixation

A combined symposium entitled “The Role of Pharmacological Agents in Fracture-Healing and Implant Fixation” was presented at the recent meeting of the Orthopaedic Research Society and the American Academy of Orthopaedic Surgeons. This symposium examined the mechanisms by which various pharmacologic agents affect bone repair and included a discussion of potential clinical applications. Because bisphosphonates reduce bone resorption while parathyroid hormone stimulates bone formation, it is possible that the concurrent administration of these two agents could increase bone density more than the use of either one alone. In two randomized trials that were recently reported in The New England Journal of Medicine, patients were given alendronate alone, parathyroid hormone alone, or both drugs22,23. In both studies, there was no evidence of synergy between the two drugs. Increases in bone mineral density were significantly greater in patients who were managed with parathyroid hormone alone as compared with alendronate alone or combination therapy. These results show that the concurrent use of alendronate attenuates the anabolic effects of parathyroid hormone.

There has been recent interest in the effect of osteoporosis drugs on fracture-healing. There is accumulating evidence that bisphosphonates delay fracture-healing. Animal studies have demonstrated the persistence of woven bone rather than progression to mature lamellar bone at the sites of healing fractures. Bisphosphonates also were found to delay healing at the site of spine fusion in a rabbit model. However, a positive effect of bisphosphonates during fracture-healing is the prevention of disuse osteoporosis. In contrast, parathyroid hormone enhances bone formation at the site of a healing fracture. Alkhiary et al. recently reported that daily systemic administration of low-dose parathyroid hormone (1-34) enhanced fracture-healing in a rat model24. The consensus opinion at the present time is that bisphosphonates should be discontinued until a fracture has healed and that parathyroid hormone may be a better option for the treatment of osteoporosis during fracture-healing. There also has been recent evidence that bisphosphonates have a positive effect in patients with osteonecrosis. In osteonecrosis, resorption may weaken the bone between the nonviable and viable bone, resulting in an increased risk of collapse.

Recent studies have also focused on the long-term effects of bisphosphonates. Bisphosphonates decrease bone resorption by inhibiting osteoclasts and delaying bone remodeling. Animal models have demonstrated that there is an accumulation of microinjury that is not remodeled. However, a recent study demonstrated that patients who were managed with alendronate for as long as ten years had sustained protection against osteoporotic fracture25. The discontinuation of alendronate resulted in the gradual loss of its effects. Additional studies are needed in order to increase our understanding of the implications of the microarchitectural changes in bone that occur in association with long-term bisphosphonate therapy and to determine the ideal duration of treatment with this agent.

Implant fixation also may be improved with use of pharmacologic agents. Recent work from Rush Medical College has demonstrated that exogenous growth factors can be roughly twofold to threefold more effective than autogenous bone-grafting in improving implant fixation. Additional studies will be necessary to determine the optimal carrier vehicle for growth factor delivery at the implant- bone interface. Currently, there is very little information regarding the comparative effectiveness of different carrier vehicles. The investigators from Rush Medical College reported alterations in gene expression at the implant-bone interface in response to exogenous TGF-[beta]2.

How Does Mechanical Load Affect Soft Tissues?

Overuse/Tendon Damage

Overuse tendon injury (tendinopathy) is a very common clinical problem. The underlying pathophysiology of repetitive stress injury to tendon is still poorly understood. Recent studies have focused on defining the mechanism of microscopic damage accumulation in tendon. Several studies presented at the recent meeting of the Orthopaedic Research Society focused on the basic mechanism of tendinosis. The most well-established animal model of overuse tendinosis involves excessive treadmill exercise in the rat. Perry et al. reported that proinflammatory pathways (identified by fivelipoxygenase-activating protein and COX-2 gene expression) and a marker of angiogenesis (Von Willebrand Factor) were elevated in this overuse model26. These alterations in gene expression were associated with detrimental effects on the histological, geometrical, and mechanical properties of the tendon. Another group studied a rabbit model of epicondylitis in which the flexor digitorum profundus tendon was repetitively stimulated against a load. There was an increase in the size and area of microtears in the tendon and increased expression of vascular endothelial growth factor in that model. These studies suggest that angiogenesis is stimulated in the early stages of tendinosis. Localized areas of cell death are seen in the later stages of repetitive stress injury to tendon. Tian et al. reported that cells that were stressed by hyperthermia and cyclic strain had increased caspase-3 activity (an established initiator of apoptosis) and DNA fragmentation, which is indic\ative of apoptosis27. In another study, collagen fibril sliding at the microstructural level was quantified with use of rat tail tendon fascicles that were marked with photobleached lines on the tendon and then examined with use of confocal microscopy while the tendon was loaded under uniaxial tensile strain28. There was substantial sliding between collagen fibrils as well as collagen fibril sliding at the cellmatrix boundary, suggesting shear strain on the cell. These results suggest a mechanism for progression of tissue damage during repetitive load.

Recent work in the area of tendon mechanobiology has focused on the role of matrix metalloproteinases. Sung and colleagues, in a series of in vitro studies of ligament cells (derived from human anterior cruciate and medial collateral ligaments) that were subjected to “injurious stretch” (12% strain), found upregulation of MMP-2 and MMP-9 gene expression29. MMP-2 plays an important role in angiogenesis, and MMP-9 is involved in ligament remodeling. These findings provide one more piece of the puzzle in understanding how load affects ligament-healing and remodeling.

In contrast to tendon overload, stress deprivation also has significant effects on soft tissues. Stress deprivation leads to upregulation of MMP-1 expression in tendon cells. Manganese superoxide dismutase is upregulated in stress-deprived tendon, and this molecule also upregulates MMP-1, thus contributing to mechanical deterioration in stress-deprived tendon. Lavagnino et al. proposed the novel concept that tendon cells may have a “mechanostat” set point to maintain homeostasis by generating and regulating internal tension in the cytoskeleton in response to changes in external strains30. They provided evidence that tendon cells may have a threshold level of strain below which gene expression (such as MMP-1 expression) is turned on. A tendon cell would be able to “recalibrate” this set point by generating internal fractional strains through the actin cytoskeleton.

Although numerous investigators have examined the in vivo effect of mechanical stimulation on ligament and tendon as well as the mechanisms of mechanotransduction in cells derived from ligament and tendon, there are very few data on the effect of mechanical load on healing at soft tissue-to-bone attachments sites. This question has considerable clinical relevance for the rehabilitation of patients following rotator cuff tendon repair and ligament reconstruction procedures (such as anterior cruciate ligament reconstruction) involving the use of a tendon graft. Thomopoulos et al., in a rat supraspinatus tendon repair model, found that immobilized shoulders demonstrated superior biomechanical properties (as demonstrated by quasilinear viscoelastic analysis), structural properties (as indicated by collagen orientation), and compositional properties (as indicated by gene expression) when compared with shoulders from animals that had been allowed normal cage activity or treadmill exercise31. These findings were contrary to expectations, given the known beneficial effect of mechanical stimulus on tendon. In animals that had been treated with exercise, healing was characterized by the formation of scar tissue with inferior material properties; in contrast, immobilization appeared to promote the expression of genes that are found at a normal insertion site. Additional studies are required to determine the appropriate postoperative loading regimen that will optimize rotator cuff tendon-tobone healing.

The Latest on Nonsteroidal Anti-Inflammatory Medications

It has been well established that nonsteroidal anti-inflammatory drugs can inhibit bone-healing. With the increasing popularity of cyclo-oxygenase-2 (COX-2) inhibitors, recent studies have examined the possibility that COX-2 inhibitors have an adverse effect on fracture-healing. Two important papers that were published in 2002 demonstrated significant impairment of fracture-healing in rats that had been given COX-2 inhibitors following the production of a closed femoral fracture and in mice that were homozygous for a null mutation in the COX-2 gene32,33. Although these studies provide compelling evidence suggesting that clinicians should consider not using COX-2 inhibitors in patients who require bone formation for fracture repair, spinal fusion, or fixation of bone-ingrowth (uncemented) prostheses, there is currently a lack of high-quality clinical data on the effect of these agents on bone-healing in humans. Furthermore, COX-2 inhibitors are effective for relieving the pain associated with inflammation after injury and surgery. How does the clinician use the basic science data to resolve this dilemma? Several studies that were presented at the recent meeting of the Orthopaedic Research Society helped to shed some light on this issue. One study involving the use of a rat femoral fracture model demonstrated that delaying treatment with the COX-2 inhibitor celecoxib until two weeks after a fracture did not delay fracture- healing, in contrast to administration of the drug immediately postoperatively or starting at seven days postoperatively34. Another study involving the use of a rat femoral fracture model demonstrated that the COX-2-induced delay in healing was reversible and that healing capacity recovered after discontinuation of treatment in animals that had been treated for as long as twenty-one days. Short- term treatment with rofecoxib (for a period of two weeks) also did not impair bone formation in a bone-harvest chamber at six weeks in a rabbit model. Currently, it appears that short-term use of these drugs is safe following skeletal surgery in otherwise healthy patients, but their use should be avoided in patients with risk factors for impaired fracture-healing, such as glucocorticoid use, metabolic bone disease, and smoking.

Recent studies also have examined the effect of nonsteroidal anti- inflammatory drugs on soft-tissue healing. Cohen et al. reported that both a nonselective nonsteroidal anti-inflammatory drug indomethacin and a COX-2 inhibitor (celecoxib) impaired healing in a rat supraspinatus tendon-to-bone repair model according to histological and biomechanical criteria35. Aspenberg et al. found that early administration of the COX-2 inhibitor parecoxib impaired Achilles tendon-healing in rats, whereas later administration had a positive effect on healing36. The authors hypothesized that inflammation is necessary for the initiation of healing (perhaps because of the production of growth and differentiation factors by inflammatory cells) but may impair late tendon remodeling. Additional studies are required before clinical recommendations can be made about the use of these drugs following tendon repair surgery.

What’s New in Spine Research

A symposium on the mechanobiological influences on intervertebral disc degeneration and repair was presented at the recent meeting of the Orthopaedic Research Society. The genetic, biochemical, mechanical, and biological mediators of intervertebral disc degeneration were discussed. Investigators are exploring the potential for cytokines to augment intervertebral disc-healing and regeneration, with the goal of developing novel treatment options, such as gene-based and cell-based therapies. Kang and colleagues at the University of Pittsburgh developed a rabbit model of gradually progressive disc degeneration and subsequently demonstrated progressive disc degeneration according to histological criteria, radiographic criteria (as seen on plain radiographs and magnetic resonance images), and molecular criteria (as demonstrated by quantitative polymerase chain reaction)37. This reproducible model of intervertebral disc degeneration was used to demonstrate the feasibility of in vivo transfer of therapeutic genes to target cells in the disc. Gene therapy potentially can be used to enhance anabolic processes (e.g., proteoglycan synthesis) and/or to inhibit catabolic processes (e.g., MMP expression) in the intervertebral disc. This animal model can also be used to test cellular therapies (e.g., those involving stem cells and transfected cells) and synthetic disc implants.

Another active area of spine research involves the examination of mechanotransduction in intervertebral disc cells. It has been established that excessive mechanical load can result in increased metalloproteinase activity and cytokine production, leading to disc degeneration and possibly stimulating back pain mechanisms. Intervertebral disc cells respond to mechanical stimulation by means of mechanoreceptors that modulate cell-matrix and cell-cell interactions, with associated changes in cell proliferation and matrix synthesis. Additional studies in this area will shed light on the mechanism of age-related disc degeneration and possibly will suggest new methods for preventing or even reversing such changes.

Tissue Engineering for Tendon

A symposium entitled “Tendon Repair and Regeneration: Challenges and Opportunities for Engineered Tissue Constructs” was presented at the recent combined meeting of the Orthopaedic Research Society and the American Academy of Orthopaedic Surgeons. This symposium defined and reviewed the design criteria for tissue-engineered tendon constructs. Tissue engineering of tendon can be broadly defined as including not only the development of replacement tissues (such as with the use of cell-scaffold techniques) but also the development of techniques to improve standard tendon repairs and graft reconstructions. Various strategies for the enhancement of tendon- healing were reviewed, including strategies involving the use of processed, naturally occurring extracellular matrices (such as porcine small intestine submucosa and processed human dermis) as scaffolds for tendon repair; cell-based strategies (for example, seeding a bioabsorbable scaffold with fibroblasts, tenocytes, or mesenchymal stem cells); and strategies involving \the use of growth factors. In the classic tissue-engineering paradigm, the cell- scaffold construct would undergo in vitro conditioning in a bioreactor, with chemical and/or mechanical factors being used to stimulate neotendon formation. The only techniques that are currently available for clinical use include the use of scaffolds and platelet concentrate gels as a source of growth factors. However, we are aware of no published studies supporting the efficacy of these approaches in clinical use.

Additional studies in this area are required to increase our understanding of the biologic response to tissue-engineered constructs and to define the optimal biomechanical parameters. It is recognized that the success of tissue-engineering will be improved by carefully defining the design parameters, which should be based on an accurate assessment of the in vivo forces and displacements that these structures will encounter following surgical implantation. Ideally, a tissueengineered construct should exceed the expected in vivo forces and also should have a stiffness that is similar to that of normal tendon. Biological/synthetic scaffolds are required that neither stress-shield the cells because they are too stiff nor create excessive deformations because they are too compliant. The scaffold also should control cell phenotype. Additional studies are needed to increase our understanding of the host tissue response to permanent, nonresorbable implants as opposed to temporary, resorbable implants. For example, it is likely that acute, subacute, and chronic inflammation in response to an implant will affect the structural and functional result.

What’s New in Biomaterials

Alternate Bearing Materials

To combat the problem of debris-mediated osteolysis, alternative materials for the bearing surfaces of total joint replacements are under continual development; the most prominent of these materials are highly cross-linked polyethylene-onmetal and alumina-on- alumina, which were approved for use in the United States in 1997 and 2002, respectively. Early retrieval analyses of highly cross- linked polyethylene components have thus far corroborated the results of simulator tests in demonstrating negligible wear rates over follow-up periods of as long as two years for both acetabular liners and tibial trays38,39. It has been well established that as the level of crosslinking increases, resistance to crack propagation decreases. However, it is not yet known what effect, if any, reduced fracture toughness will have on implant failure modes and longevity40. Data from hip-simulator experiments, presented at the recent annual meeting of the Orthopaedic Research Society, demonstrated more extensive impingement-related damage in elevated cross-linked polyethylene liners than in conventional polyethylene liners41. Isolated cases of liner fracture also have been recently reported to the Food and Drug Administration (Manufacturer and User Facility Device Experience database), underscoring the need for closely monitored follow-up performance analysis.

The reduced biological activity of alumina debris as compared with polyethylene debris continues to be shown in cell-culture models42; however, the risk of alumina liner fracture remains a continuing concern. Although liner fractures have been reported to the Food and Drug Administration, the cause of the fractures (for example, malalignment or poor surgical technique) and the rate of fractures as a percentage of the number of implanted liners are unknown. There is a pressing need to test the behavior of ceramic liners under clinically relevant loading conditions that are likely to cause implant fracture (for example, under impingement or edge- loading).

Alternate Implants

Total disc replacements for the spine are designed to relieve pain while restoring joint mobility to the affected spinal segment. The implants that are currently approved for use in the United States utilize a metal-on-polyethylene articulation to completely replace the disc. In 2003, the preliminary data from prospective, randomized studies comparing the performance of two total disc- replacement designs with that of spine fusion were reported43,44. While both disc-replacement designs were associated with minimal complications at the time of early follow-up, long-term follow-up will be required. Useful information about failure modes and complications may emerge from Europe, where total disc replacements have been used for more than twenty years. In an extensive study of complications following metal-on-polyethylene total disc replacement, van Ooij et al. reported complications including degeneration at an adjacent level and implant subsidence, at a mean of fifty-three months after implantation45. Although polyethylene wear was evident in one patient, evidence of osteolysis was not reported.

Hydrogels are a soft, porous-permeable family of polymers with high water contents and mechanical properties that can be varied by an order of magnitude by altering the composition (i.e., the polymer blend or cross-linking) and/or the structure (i.e., the geometry or porosity) of the material. The use of synthetic, nondegradable hydrogels as load-bearing orthopaedic implants is a recent development in orthopaedic research. Unlike traditional implants, in which the joint loads are mainly transferred through the construct to the surrounding tissues, hydrogel implants are being developed predominantly to share load with adjacent tissues. Applications under development, although not yet in clinical trials, include meniscus replacements, nucleus pulposus replacements, and hemiarthroplasties. Kobayashi et al. demonstrated the chondroprotective ability of a hydrogel (polyvinyl-alcohol) meniscus replacement in a rabbit model in which knees that were treated with an artificial meniscus were compared with meniscectomized knees46. The same group of investigators developed a composite osteochondral device consisting of an injection-molded hydrogel that is infiltrated into a titanium mesh. To date, the implant has been used as a femoral head hemiarthroplasty and as an intervertebral disc replacement in a canine model47. In both cases, the device achieved firm attachment to the underlying bone and no pathological changes were found adjacent to the device as long as one year postoperatively.

Synthetic Biologically Active Materials

A relatively new concept in tissue engineering is to integrate bio-active molecules into scaffolds to help to guide tissue regeneration. Pratt et al. developed a polymer network into which various peptides with specific regulatory functions were linked48. Specifically, adhesion peptides allowed the cells to attach to and migrate through the scaffold, MMPsensitive sites allowed for the enzymatic remodeling of the scaffold by invading cells, and growth factors were incorporated so that, as the cells remodeled the matrix, the growth factors were mobilized to enhance the remodeling process. The scaffold facilitated osteoblast ingrowth in cell cultures and bone-tissue generation in a rat cranial defect model. However, the mechanical integrity of the scaffold immediately upon insertion and during the remodeling process was not quantified; hence, its ability to augment tissue repair in a load-bearing environment (e.g., close to the surface of a joint) is unknown.

Manufacturing of Scaffolds

Engineering the geometric features of a scaffold at the nanoscale (10^sup -9^ m) and microscale (10^sup -6^ m) enables control over cellular infiltration, cellular adhesion, and degradation characteristics. Tan and Saltzman adopted microfabrication photolithography processes that are used in the semiconductor industry and combined them with deposition techniques mimicking the natural formation of tissues to impose controlled nanosized structural features on a micrometer-sized matrix49. Briefly, radiation was transmitted through a micropatterned mask onto a radiation-sensitive substrate, thus transferring the micropattern of the mask to the substrate. Ion etching then transferred the pattern to silicon wafers to generate the microsized geometries. Tan and Saltzman then chemically altered the surface of the micropatterned material with surface moieties to allow biological molecules (in this case, nanostructured hydroxyapatite) to attach to the surface. This last step of the novel process superimposed the nanosized geometry onto the microsized feature of the scaffold for the purposes of engineering bone tissue around the scaffold. In a modified photolithographic procedure, Luo and Shoichet chemically modified thermosensitive agarose hydrogels with photosensitive molecules50. When subjected to ultraviolet light, the photosensitive molecules were cleaved in order to facilitate the attachment of biomolecules. By selectively exposing areas of the polymer to ultraviolet light, three-dimensional biochemical channels were created to guide the direction of tissue growth.

Using another novel manufacturing approach, Luu et al. incorporated DNA plasmid into a structurally stable polymer scaffold with use of electrospinning techniques51. Briefly, in electrospinning, a liquid polymer jet is forced through an electrically charged nozzle. The electrical force overcomes the surface tension of the polymer jet, causing it to split into multiple filaments. The filaments are ejected toward a collecting screen, creating a complex pattern of submicrometer-sized polymer fibers. Luu et al. successfully incorporated DNA plasmid into the liquid polymer jet and generated a DNA-polymer interconnected porous network with tensile properties similar to that of cartilage, resulting in controlled release of DNA from the scaffold.

Nanotnechanics

A workshop on techniques used to investigate the mechanics of biological tissues at the nanoscale was presented at the recent meeting of the Orthopaedic Research Society. Among the techniques described were a\tomic force microscopy, highresolution spectroscopy, nanoindentation, Raman spectroscopy, and the use of optical tweezers. Although none of these techniques are new, their combined application for the purposes of understanding the structural, molecular, and mechanical properties of musculoskeletal tissues at the nanoscale is novel.

Atomic force microscopy can be used to generate topographical information about a material. The technique involves the use of a nanosized probe mounted on a microsized cantilever. The apparatus is programmed to keep the vertical displacement of the cantilever constant so that, as the probe moves across a textured surface, the height correction that it must implement creates a topographical map of the surface. The probe can be used to peel away surface layers, allowing for a three-dimensional structural characterization of a material-for example, the structure of a fibrillar network52, the structure of macromolecules53, and the shape distribution of mileralite sites. In high-resolution spectroscopy, the same probe is used, but in this technique the probe is moved at a constant rate toward and then away from the surface. The cantilever deflects in response to surface interactions. With knowledge of the spring constant of the cantilever and the deflection of the cantilever, the force generated by the interaction can be computed. The probe can be functionalized with ligands and receptors and more complex interactions can be investigated. High-resolution spectroscopy can be used to provide valuable information about molecular interactions in healthy, diseased, and surgically manipulated tissues.

Nanoindentation also involves the use of a probe, but in this case the probe deforms the surface of the material; the applied force and the material deformation are recorded, and from these data the hardness (resistance to indentation) and the elastic modulus of the material are computed. Nanoindentation has been used to measure the mechanical properties of trabeculae of bone and to quantify how systemic treatments for diseases, such as osteoarthritis, affect those properties54. More recently, nanoindentation methods have been developed to quantify the properties of soft hydrated tissues55. For example, Alien and Mao combined atomic force microscopy with nanoindentation to explore the regional variation in structural and mechanical properties in the interterritorial and pericellular matrices of cartilage in order to understand the effect of changes in these properties on the function of cartilage56.

Optical tweezers are used to measure the mechanical properties of components at a smaller scale than that of nanoindentation. To measure the tensile strength of a molecule, for example, the molecule is attached to a small bead at one end and to a much larger bead at the other end. The small bead remains fixed in place, while the larger bead is fixed between parallel coverslips. The coverslips are attached to an XY piezostage, so that when the stage is moved, the molecule is stretched. The force exerted by the molecule on the bead is measured and the stiffness can be calculated. Recently, optical tweezers were used by Huang et al. to elucidate the mechanics of chondrocyte adhesion by measuring cell plasma membrane tether formation57. While each of these processes provides important information about the basic physical, chemical, and mechanical structure of molecules and matrices in biological tissues, it is the combined use of these processes that will advance our understanding of the interactions between cells and their environment and the effect that disease has on these interactions. These novel tools also will prove to be useful for the evaluation of tissueengineered structures.

Upcoming Meetings and Opportunities Related to Orthopaedic Research

The pace of discovery has continued to accelerate as research techniques and methods have become more sophisticated. The challenge for the research community is to translate important research findings to the clinic as rapidly as possible. This points out the importance of training and supporting orthopaedic surgeon- scientists. Several programs have been developed by the American Academy of Orthopaedic Surgeons (AAOS) and the Orthopaedic Research and Education Foundation (OREF) to foster and support orthopaedic surgeon-scientists. The OREF has developed a Clinician Scientist Award that provides salary support up to $100,000 per year for up to three years to an orthopaedic surgeon to spend 40% of his or her time in research. The AAOS and the OREF have developed the Clinician Scientist Development Program (CSDP), which targets residents in their PGY3 and PGY4 training years who have the potential to become clinician-scientists. The program is designed to provide prospective clinician-scientists with an extensive orientation to orthopaedic research and opportunities in the field. The AAOS and the OREF also jointly sponsor a health services research fellowship. The goal of this program is to encourage the development of research skills necessary to carry out health services and outcomes research. The candidate is encouraged to earn a public health degree.

The Orthopaedic Research Society web site has a listing of scientists who have agreed to serve as mentors for young scientists (www.ors.org/mentoring/mentor.asp). This web site helps to match students with a mentor who has agreed to review grants and manuscripts or to host a young scientist for a period of time to learn new techniques. The Orthopaedic Research Society also continues to offer a Traveling Fellowship award. This fellowship provides funding for an orthopaedic researcher (clinician or basic scientist) who is under the age of forty years to visit one or several laboratories to learn new research techniques and to establish research collaborations. Further information about the Traveling Fellowship is available through the Orthopaedic Research Society. Opportunities for research fellowships are also listed on the web site of the International Center for Orthopaedic Education (www.icoe.aoassn.org), which is sponsored by the American Orthopaedic Association.

Several upcoming meetings over the next year will provide timely updates on the latest findings, trends, and techniques in orthopaedic research for the clinician. The American, Canadian, European, and Japanese Orthopaedic Research Societies will hold the Fifth Combined Meeting in Banff, Alberta, Canada, from October 10 through 13, 2004. The International Cartilage Repair Society holds an annual meeting each spring at which the latest developments in the basic and clinical aspects of cartilage repair are presented. The American Society for Bone and Mineral Research (ASBMR) will hold its Twenty-sixth Annual Meeting on October 1 through 5 in Seattle, Washington (www.asbmr.org). The annual Grant Writing Workshop will be held at the offices of the American Academy of Orthopaedic Surgeons in Rosemont, Illinois, in the spring of 2005. This meeting is aimed at assisting orthopaedic surgeons who are writing NIH RO-1 research grant proposals. The Annual Meeting of the Orthopaedic Research Society takes place just before the Annual Meeting of the American Academy of Orthopaedic Surgeons. The Fifth Annual International Symposium on Tendons and Ligaments will be held one day before the Orthopaedic Research Society Annual Meeting. Information about this meeting is available from Savio Woo, PhD, at the University of Pittsburgh.

NOTE: The authors thank the following experts in various fields of orthopaedic research for providing input and information for this review: Howard S. An, MD (Chicago, Illinois), intervertebral disc; David Butler, PhD (Cincinnati, Ohio), tendon tissue engineering; Lou Gerstenfeld, PhD (Boston, Massachusetts), pharmacological agents in fracture-healing; Joseph P. Iannotti, MD, PhD (Cleveland, Ohio), tendon tissue engineering; James Kang, MD (Pittsburgh, Pennsylvania), intervertebral disc; Joe Lane, MD (New York City), pharmacological agents in fracture-healing; Jeff Lotz, PhD (San Francisco, California), intervertebral disc; Rick Sumner, PhD (Chicago, Illinois), pharmacological agents implant fixation; Charles Turner, PhD (Indianapolis, Indiana), genetics and mechanical loading in bone.

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BY SCOTT A. RODEO, MD, SUZANNE A. MAHER, PHD, AND CHISA HIDAKA, MD

Scott A. Rodeo, MD

Suzanne A. Maher, PhD

Chisa Hidaka, MD

Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021.

E-mail address for S.A. Rodeo: rodeos@hss.edu

The authors did not receive grants or outside funding in support of their research or preparation of this manuscript. They did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

Copyright Journal of Bone and Joint Surgery, Inc. Sep 2004