Extract
Progressive advances continue to be made in several areas of orthopaedic research. The major focus is on biologic solutions to musculoskeletal injury and degenerative joint disease. At the recent meeting of the Orthopaedic Research Society, studies and workshops were presented on stem cells, growth factors, the genetic basis of disease, and tissue-engineering. Investigations in these areas have the potential to improve current treatments and to lead to the development of novel therapies and implants. There is continuing progress in the understanding of the genetic basis of diseases, such as degenerative joint disease, and the biologic response to injury. Biomechanics and engineering also continue to be important areas of investigation. In particular, several laboratories are investigating the effect of mechanical stimuli on musculoskeletal development, chondrocyte metabolism, skeletal repair, and tissue-engineered constructs.
Progressive advances continue to be made in several areas of orthopaedic research. The major focus is on biologic solutions to musculoskeletal injury and degenerative joint disease. At the recent meeting of the Orthopaedic Research Society, studies and workshops were presented on stem cells, growth factors, the genetic basis of disease, and tissue-engineering. Investigations in these areas have the potential to improve current treatments and to lead to the development of novel therapies and implants. There is continuing progress in the understanding of the genetic basis of diseases, such as degenerative joint disease, and the biologic response to injury. Biomechanics and engineering also continue to be important areas of investigation. In particular, several laboratories are investigating the effect of mechanical stimuli on musculoskeletal development, chondrocyte metabolism, skeletal repair, and tissue-engineered constructs.
This year's combined Orthopaedic Research Society/American Academy of Orthopaedic Surgeons symposium focused on the clinical applications of growth factors. Growth factors are secreted proteins that act on a target cell or cells to carry out functions such as cell division, cell differentiation, and matrix synthesis. Binding of a growth factor to its receptor activates a signal transduction system, which eventually effects nuclear gene expression. Transmission of the signal from the ligand-receptor interaction involves transcription factors, which are intracellular proteins that are activated by the signaling pathway and that bind to nuclear DNA, ultimately effecting gene expression. The ability of a growth factor to induce a biologic effect depends on a carrier system to deliver the factor to the appropriate site in a biologically relevant concentration. An active area of investigation is the identification of effective carrier vehicles for growth factor delivery. Recently, researchers have focused on the potential for gene therapy techniques to induce a cell to produce the desired factor. Both viral vectors (e.g., retrovirus, adenovirus, and lentivirus) and nonviral vectors (e.g., liposomes, naked DNA, and gene-activated matrices) have been used to transfer genetic information to a cell.
The major focus of growth factor research has been on skeletal repair (e.g., fracture-healing, revision arthroplasties for which structural bone support is required, and spinal fusion). Extensive preclinical testing of bone morphogenetic proteins (BMPs) has resulted in their approval by the Food and Drug Administration (FDA) for clinical application. The genes encoding BMP-2 and BMP-7 have been isolated and now allow unlimited production of these proteins. Osteogenic protein-1 (OP-1, also known as BMP-7) was the first osteoinductive factor to be approved by the FDA. It received a Humanitarian Device Exemption for use in the treatment of recalcitrant long-bone nonunions for which the use of autograft is unfeasible and alternative treatments have failed. The OP-1 implant has also been approved for use in Australia and the European Union for specific trauma-related indications. In a recent report, sixty-three tibial nonunions that were treated with an intramedullary rod and recombinant human OP-1 (rhOP-1) were compared with sixty-one tibial nonunions that were treated with an intramedullary rod and autogenous bone graft
1 . The authors reported no differences between the two groups with regard to the clinical outcome or the rate of fracture union.
Recombinant human bone morphogenetic protein-2 (rhBMP-2) has also become available for use in the United States. FDA approval was granted on July 2, 2002, for the use of rhBMP-2 with a collagen-sponge carrier and a titanium cage for single-level interbody spinal fusion. Approval for the use of rhBMP-2 for spine fusion has also been granted in Canada. rhBMP-2 has also been reported to be effective for the treatment of open tibial fractures. In a recent prospective, randomized, controlled, single-blind study, 450 patients with an open tibial fracture were randomized to receive either the standard of care (intramedullary nail fixation and routine soft-tissue management), the standard of care and an implant containing 0.75 mg/mL of rhBMP-2 (total dose, 6 mg), or the standard of care and an implant containing 1.50 mg/mL of rhBMP-2 (total dose, 12 mg)
2 . The rhBMP-2 implant in a collagen-sponge carrier was placed over the fracture at the time of wound closure. The group treated with 1.50 mg/mL of rhBMP-2 had a 44% reduction in the risk of failure (defined as secondary intervention because of delayed union) (p = 0.0005) and significantly faster fracture-healing (p = 0.0022) than did the control patients. The group treated with 1.50 mg/mL of rhBMP-2 also had significantly fewer instances of hardware failure, fewer infections, and faster wound-healing. The authors concluded that the implant containing 1.50 mg/mL of rhBMP-2 was significantly superior to the standard of care in patients with an open fracture of the tibia.
rhBMP-2 on an absorbable collagen-sponge carrier was approved in 2002 by the European Union for use in Europe for the "treatment of acute tibia fractures in adults, as an adjunct to standard care using open fracture reduction and intramedullary nail fixation" (www.eudra.org/humandocs/humans/EPAR/inductos/inductos.htm). The European product is called InductOs (Medtronic, Minneapolis, Minnesota). On November 21, 2002, an FDA Advisory Panel voted 6 to 1 in favor of approval of rhBMP-2 for use in the United States for the treatment of open tibial fractures. A final FDA approval decision is pending at the time of this writing.
Parenteral administration of growth factors is also currently under investigation. Systemic intermittent dosing of parathyroid hormone (PTH) has an anabolic effect on bone. Parenteral administration of PTH or an analog has been shown to increase both bone formation and bone mass. A recent study demonstrated that patients with postmenopausal osteoporosis who had been treated with parathyroid hormone (1-34) had increased vertebral, femoral, and total-body bone-mineral density and a decreased risk of vertebral and nonvertebral fractures
3 .
Growth factors also hold promise for soft-tissue healing. Recent work has focused on identifying the most effective delivery mechanisms. Although direct delivery of a protein to a repair site has been shown to be effective in some models, there is concern that the protein of interest may not be available in a biologically relevant concentration at the appropriate times. Gene therapy techniques may prove useful for the delivery of growth factors and other biologically active molecules. Successful gene transfer to ligament, tendon, cartilage, and meniscus has been demonstrated with use of different gene therapy approaches (direct injection of adenoviral particles, injection of cells that have been transduced ex vivo, and nonviral gene delivery)
4 . Recent investigations have demonstrated positive biologic effects of the transgene. For example, Lou et al. showed that BMP-12 gene transfer into a complete tendon laceration in a chicken model resulted in a twofold increase in the tensile strength and stiffness of repaired tendons
5 . Martinek et al. demonstrated that BMP-2 gene transfer improved the integration of a semitendinosus tendon graft at the tendon-bone interface after reconstruction of the anterior cruciate ligament in rabbits
v . Those authors reported reestablishment of a normal, direct insertion site as well as significantly increased stiffness and ultimate load-to-failure in association with the BMP-2-transduced grafts (p < 0.05).
New insights into the role of cytokines in tendon-healing have been gained with use of transgenic mouse models. Lin et al. studied the healing of a patellar tendon defect in interleukin-6 (IL-6) knockout mice
7 . They found increased cross-sectional area (indicative of a greater inflammatory response) and a trend toward lower material properties in the IL-6 knockout animals. Since IL-6 is a pro-inflammatory mediator, it was surprising that there would be an apparent increase in inflammation in the IL-6 knockout mice, and it is likely that other pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-a) are upregulated in the absence of IL-6. Another study was performed to evaluate Achilles tendon-healing in mice lacking the gene for inducible nitric oxide synthase (iNOS) because previous work had demonstrated that NOS inhibition impaired tendon-healing. There was no difference in healing in the iNOS knockout animals compared with controls, but systemic NOS inhibition (with use of aminoguanidine) did result in decreased cross-sectional area, suggesting that the iNOS gene is not solely responsible for the beneficial effect of nitric oxide on tendon-healing. These two studies illustrate the complexity of tendon-healing.
Mechanobiology in Musculoskeletal Development
Ideally, the healing of musculoskeletal tissues should recapitulate the process that occurs during embryonic development. Improved understanding of the basic processes that govern the development of musculoskeletal tissues can provide important insights for tissue repair and tissue-engineering. Recent studies have demonstrated that musculoskeletal development is influenced both by intrinsic genetic factors and by the mechanical loading environment. A workshop at this year's meeting of the Orthopaedic Research Society reviewed the effect of mechanical load on gene expression during embryogenesis. The developing skeleton is subjected to complicated distributions of physical deformations (strains) and pressures. These mechanical stimuli regulate morphogenesis throughout development, with specific effects on growth rates, the direction of tissue growth, and differentiation. Skeletal growth generates strains and pressures that in turn influence gene expression. For example, if growth is more rapid on one side of a developing tissue than on the other (as would be the case if there was faster growth on the volar surface than on the dorsal surface of a developing limb-bud), the faster-growing tissue will experience pressure as the slower-growing tissue acts as a constraint. The slower-growing tissue will experience tension as the faster-growing tissue imparts a stretching force. These different forces (tension and compression) are likely to induce differential gene expression. Intense study is being focused on understanding the response pathways and cellular adaptive responses to mechanical signals.
Noninvasive experimental models have been developed to study adaptation of bone to mechanical stimuli. Fritton et al. modified the ulnar compression model for use in the mouse, allowing for the control of the rate, magnitude, and duration of loading
8 . Transgenic mouse models also continue to provide a powerful tool for understanding the genetic factors that control skeletal development and adaptation. For example, Ishijima et al. recently reported that the osteopontin-knockout mouse does not lose bone during hindlimb suspension, suggesting that osteopontin is involved in bone-remodeling
9 . In a unique experiment, Rodriguez et al. developed an in vitro system to apply controlled motion to an isolated chick embryo hindlimb in tissue culture to study joint morphogenesis
10 . They demonstrated that hindlimbs that were exposed to the dynamic conditions had better joint differentiation and higher glycosaminoglycan in the developing chondral surfaces. This model will be useful for further controlled studies on joint development.
Recent studies have focused on identifying the signals that are triggered in connective-tissue cells by mechanical stress and how such stimuli effect extracellular matrix protein gene expression. Integrins, transmembrane adhesion and signaling receptors that physically link the extracellular matrix to the cytoskeleton, appear to play an important role in transducing mechanical signals. Integrins appear to function by triggering MAP kinase and NF-kappaB pathways in response to changes in mechanical stress
11 . The cellular mechanotransduction pathway may activate transcription factors. One important transcription factor for mechanotransduction is EGR-1 because its mRNA is immediately upregulated after application of tensile stress to cultured cells. The end result of mechanotransduction is a change in gene expression, probably due to binding of the transcription factor to a mechanoresponsive region of the extracellular matrix gene promoter. For example, both the tenascin-C and the collagen XII gene promoters contain sequences that respond to static stretch.
An intact actin cytoskeleton is critical for this mechanotransduction process. It appears that fibroblasts need to maintain a certain amount of internal cytoskeletal tension in order to be able to sense mechanical stress from extracellular matrix strain. For example, disruption of the cytoskeleton suppresses induction of the tenascin-C gene by cyclic stretch. It is known that cyclic strain is associated with an activation of stress-activated protein kinases, and Arnoczky et al. recently showed that cyclic strain resulted in activation of c-Jun N-terminal kinase (JNK) in canine patellar tendon cells
12 . Arnoczky et al. also recently showed a correlation between tendon strain and cell nucleus deformation in statically loaded tendons, suggesting that deformation may play an important role in the mechanical signal transduction pathway of this tissue
13 .
Very recently published data provide further insight into the specific candidate molecules that are involved in mechanotransduction. Fluck et al. reported that the mechanodependent production of collagen XII and fibronectin requires separate pathways
14 . Tensile stress-dependent collagen XII production was mediated by the focal adhesion kinase (FAK)-ERK1/2 (a MAP kinase) pathway, a genistein-sensitive tyrosine kinase, and a classical/novel protein kinase C (PKC). In contrast, fibronectin induction was regulated by a different PKC-dependent pathway. The MAP kinases ERK1/2 acted at focal adhesion complexes when integrins were exposed to mechanical stress, and protein kinase C regulated cytoskeletal function. That study
14 illustrates the complexity of, and the diversity in, the intracellular signaling pathways. Furthermore, it is likely that different types of mechanical stress (e.g., tension, compression, and shear stress) induce different cellular responses via distinct signaling pathways. Further studies are required to define these diverse pathways.
Tendon Development
Recent studies have identified molecular signals that govern tendon and tendon insertion site development. Transcription factors play important roles in the process of determination and differentiation of tendon cells. Scleraxis is a transcription factor that is expressed in mesenchymal progenitors that later form connective tissues, including tendons and tendon insertion sites. Sox9 is a transcription factor that is expressed at high levels in chondrocytes. These two transcription factors appear to regulate the fate of cells that interact with each other at the interface between the skeleton and tendon and to determine the divergence of their differentiation pathways. Early in development, scleraxis and Sox9 are expressed in similar areas, but, as development proceeds, scleraxis transcripts are more restricted to tendons while Sox9 expression is confined within the early skeletal primordia
15 . Scleraxis expression is regulated by BMP signaling. Fibroblast growth factors also play a role in tendon development. Fibroblast growth factor-8 (FGF-8), which is a secreted growth factor that is involved in the initiation, outgrowth, and patterning of vertebrate limbs, is expressed in tendons during limb development. FGF-4 regulates the expression of tenascin and scleraxis in tendon
16 . Transcripts for teneurin-2, a member of a novel family of transmembrane proteins, also appear transiently at sites of tendon development
17 . Teneurin-2 expression patterns are similar to those of FGF-8. Insight into the developmental biology of tendon will be applied to the tissue-engineering of tendon and will provide a basis for new tendon-healing strategies.
The importance of growth/differentiation factor-5 (GDF-5) in tendon development was reported by Mikic et al.
18 . They found that tendons from GDF-5-deficient mice exhibited altered composition and mechanical properties when compared with heterozygous controls. GDF-5-deficient Achilles tendons were structurally weaker (p < 0.0001) than controls and contained significantly less collagen (p < 0.004) per microgram of DNA when compared with controls. Finally, Smad-signaling pathways may be involved in tendon and ligament formation. In a study presented at the recent Annual Meeting of the Orthopaedic Research Society, Gazit et al. reported that Smad8-transfected cells implanted in mice led to neoligament formation as demonstrated with use of histologic and molecular criteria
19 .
Genetic Aspects of Bone Strength
The increasing number of mouse mutants (knockins, knockouts, and so on) has created the need for multidisciplinary descriptions of skeletal phenotypes. A recent editorial in the
Journal of Orthopaedic Research presented guidelines for describing mouse skeletal phenotypes
20 . It is evident that geometric, radiographic, histologic, and mechanical property data are all relevant to skeletal phenotype. Four data categories should be included: (1) whole animal characteristics (i.e., body mass, length), (2) skeletal morphology and mineral properties (i.e., cross-sectional area, mineral content), (3) cell and matrix features (i.e., histomorphometric indices of bone formation), and (4) mechanical properties (i.e., modulus of elasticity). Adoption of these guidelines will improve the ability to compare results between studies.
There has been sustained interest within the research community in understanding the factors that determine skeletal integrity. In particular, investigators are focusing on the effect of the absence or overexpression of a gene product on bone strength. The structural properties of bone are determined by measuring structural stiffness and structural strength. These structural properties are determined by the tissue material (i.e., mineral and matrix proteins) and the tissue geometry (i.e., shape and cross-sectional area). Both material and geometric properties are required to assess the structural integrity of bone. Bone material properties are determined by the composition of the material (i.e., collagen and mineral content) and the organization of the material (i.e., osteon assembly and collagen cross-linking). Understanding bone strength ideally requires an assessment of structural, material, and geometric properties. Mineral density appears to play an important role in determining the structural stiffness and structural strength of bone. However, bone-mineral density by itself is not sufficient for predicting bone strength. For example, Lochmuller et al. recently evaluated the geometric and densitometric properties of cortical and trabecular bone in cadaveric femora with use of peripheral quantitative computed tomography and correlated these measurements with mechanical failure loads of the proximal part of the femur
21 . In that study, both geometric cortical measurements and density contributed independently to femoral strength.
A recent exciting discovery is the finding that mutations in the gene for low-density lipoprotein receptor-related protein 5 (LRP5), which acts in the Wnt signaling pathway, are associated with increased bone density
22 . This association was discovered by means of genetic and biochemical analyses of a kindred with an autosomal dominant syndrome characterized by high bone density. The mechanism is the result of increased signaling through the Wnt pathway due to loss of function of the protein Dickkopf-1 (Dkk-1), which normally inhibits Wnt signaling. These findings suggest that manipulation of Dkk signaling may be a potential target for the prevention or treatment of osteoporosis. However, consideration of the factors that control skeletal integrity makes it evident that there is not likely to be a single gene that determines bone strength. Rather, both genetic and mechanical factors influence bone geometry and material properties, which in turn determine structural behavior.
Recent studies have suggested that various toxic environmental agents have an effect on bone metabolism. Cadmium exposure and bone-mineral density were measured in individuals residing near a cadmium smelter in southeast China
23 . The authors reported a dose-effect relationship between cadmium dose and bone-mineral density and concluded that environmental exposure to cadmium is associated with an increased loss of bone-mineral density in both males and females, leading to osteoporosis and an increased risk of fractures.
Another recent study examined the effect of environmental toxins found in the tar fraction of cigarette smoke and in urban air pollution on bone metabolism
24 . That study demonstrated that the polycyclic aromatic hydrocarbons found in cigarette smoke caused a loss of bone mass and bone strength in rats, possibly through an increase in bone turnover. Previous studies have also demonstrated that lead adversely affects the function of growth plate chondrocytes, osteoblasts, and osteoclasts. Lead is stored in bone, and age-related increases in skeletal turnover result in lead release, especially in postmenopausal females. Because of its toxic effects on both osteoclasts and osteoblasts, increased serum levels of lead may exacerbate osteoporosis. Further epidemiological studies are required to fully understand the effects of various environmental agents on bone metabolism.
Investigations continue into ways to improve the wear characteristics of the polyethylene that is used for arthroplasty. New processing and sterilization protocols are being evaluated. Highly cross-linked polyethylenes were first used for total hip arthroplasty in 1998 and were first used for total knee arthroplasty in 2001. Recent work has demonstrated that thermal processing (annealing and remelting) significantly (p < 0.05) impacts the crystallinity, and hence the mechanical behavior, of highly cross-linked ultra-high molecular weight polyethylenes (UHMWPE)
25 . Thermal treatment of irradiated polyethylene can reduce the concentration of free radicals that result from irradiation, reducing oxidative degradation. Cross-linking (governed by the gamma radiation dose) and crystallinity (governed by the thermal processing) were found to be useful predictors of the mechanical behavior of UHMWPE. However, a high degree of cross-linking (with radiation doses of >100 kGy) impaired the fracture resistance. This indicates that a high degree of cross-linking is less desirable for orthopaedic implants that are used for high-stress applications, such as tibial inserts used for total knee arthroplasty. Recent studies have demonstrated that a low degree of cross-linking may be beneficial for resistance to delamination and catastrophic failure
26 . Evaluation of a highly cross-linked polyethylene tibial knee insert with use of an aggressive in vitro protocol showed no subsurface cracking or delamination at 500,000 cycles
27 . Clinical studies will be critical to determine if these new polyethylenes improve the wear characteristics of total knee implants. Encouraging short-term clinical results were noted in association with highly cross-linked UHMWPE total hip implants during the past year, but the findings have not yet been published. At the recent meeting of the Orthopaedic Research Society, Nivbrant et al. reported an 85% reduction in wear rates for highly cross-linked polyethylene cups as compared with standard polyethylene cups
28 . Only long-term clinical studies will determine if these materials will improve wear rates and decrease the prevalence of osteolysis.
Progress continues to be made toward successful engineering of bone and cartilage. The basic paradigm for tissue-engineering has been to seed cells on a synthetic, resorbable scaffold. It has become evident that cells respond differently on various polymers. Recent studies have focused on understanding cell behavior (i.e., adhesion, proliferation, and differentiation) on different types of scaffolds. The behavior of bovine articular chondrocytes on polylactic acid/polyglycolic acid (PLA/PGA) composites was recently reported
29 . As the concentration of PLA increased, cell seeding efficiency decreased and cell proliferation was slower. Additionally, the compressive modulus of scaffolds increased linearly with the addition of PLA, and the degradation time of these scaffolds also increased as the concentration of PLA increased. Effective cartilage formation has been found with use of PLA/PGA scaffolds in vitro and in athymic animals. However, in immunocompetent animals, PGA scaffolds elicit an inflammatory reaction. The resultant tissue formation in immunocompetent animals is a mixture of cartilage and fibrous tissue. Because of this limitation, other biomaterials have been tested for cartilage tissue-engineering.
Polymer hydrogels have been shown to be effective carriers for chondrocytes for cartilage tissue-engineering. Chang et al. described an effective technique in which suspensions of chondrocytes in 2% alginate are gelled by mixing with CaSO4
30 . This suspension can be injected into a specifically-shaped mold. The particular chemistry of 2% alginate mixed with CaSO4 allows the material to be injected into a mold before the cross-linking process results in a solid shape. The hydrogel scaffold promotes the chondrocyte phenotype, maintains cell viability, and supports proteoglycan and collagen production, resulting in progressive cartilage formation after implantation. This injection-molding approach allows for the production of precisely shaped cartilage implants, which could have applications for chondral defect resurfacing. The injection-molding technique with alginate also allows much more rapid seeding of the scaffold than is possible with use of PGA-based scaffolds, and it also allows high-density cell seeding.
Improved understanding of cell-material interactions will likely demonstrate that specific cell types behave differently on different scaffold materials. This information may point to a composite approach to tissue-engineering in which different types of scaffolds are used for specific cell types. For example, investigators have created tissue-engineered composites of anulus fibrosis and nucleus pulposus for intervertebral disc replacement with use of a PLA/PGA scaffold for annular cells and a 2% alginate scaffold for nucleus cells. Because many musculoskeletal structures are complex and contain different types of tissue, such a composite approach holds great promise for tissue-engineering.
Recent studies have focused on improved methods for the detection of early matrix changes in osteoarthritis. Magnetic resonance imaging continues to be the gold standard for the noninvasive evaluation of the musculoskeletal soft tissues. Current studies have focused on improving the ability to evaluate the architectural structure and biochemical composition of the extracellular matrix. In particular, information about the water, collagen, and glycosaminoglycan components of hyaline cartilage can be provided by current magnetic resonance imaging techniques. The content, orientation, and structure of collagen are the principal determinants of T2 relaxation in cartilage. Focal changes in T2 can be monitored over time or following intervention to monitor matrix damage. Information about glycosaminoglycan content can be provided with use of the contrast agent gadolinium diethylene triamine penta-acetic acid (Gd-DTPA
2- ), which is a negatively-charged molecule and thus distributes into cartilage in inverse proportion to the glycosaminoglycan content
31 . Measurement of the glycosaminoglycan concentration with use of this technique is referred to as delayed Gadolinium Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC). The dGEMRIC technique holds promise for measuring changes in cartilage glycosaminoglycan concentration during disease progression or following surgical intervention; however, further validation of the technique is required before widespread in vivo clinical application can be recommended. Diffusion of Gd-DTPA
2- into cartilage has been studied in vitro; however, further studies are required to determine the kinetics of Gd-DTPA
2- diffusion in vivo as diffusion may not be solely a function of the fixed-charge density of the cartilage. For example, further studies are required to understand the diffusion kinetics of Gd-DTPA
2- across a periosteal membrane and how an intra-articular effusion (potentially containing degradative enzymes) may affect this process.
Advanced digital post-processing techniques that allow quantitation of magnetic resonance imaging data have been described recently
32 . These techniques provide quantitative information about cartilage volume, thickness, and surface area. Accurate measurement of the regional three-dimensional signal intensity distribution in articular cartilage is also possible with these post-processing techniques. The precision and reproducibility of these measurements have been demonstrated
32 . This technique has been used to measure in situ cartilage deformation and recovery following compression loading of the joint surface (i.e., during exercise). These image-processing techniques also can be used to obtain quantitative information about joint surface size, surface curvature, and joint incongruity. Quantitative analyses of magnetization transfer and proton density also appear to be promising for the evaluation of articular cartilage as they are related to the deformational behavior of cartilage. New computed tomographic techniques also allow for the precise measurement of cartilage thickness. The technique of multi-detector computed tomography (MDCT) allows slice thicknesses as small as 0.5 mm, with excellent spatial resolution. A recent report at the Annual Meeting of the Orthopaedic Research Society demonstrated precise measurement of articular cartilage thickness with use of air-injection, contrast-enhanced MDCT
33 . In summary, new magnetic resonance imaging techniques (including indices of cartilage volume, dGEMRIC, and T2) as well as improved quantitation with post-processing allow monitoring of the anatomic, biochemical, and biomechanical properties of cartilage. These imaging modalities will allow for the detection and quantification of early degenerative processes in cartilage.
Ultra-high resolution cartilage imaging is now possible with use of high-field-strength micromagnetic resonance imaging, which allows for the imaging of small (ex vivo) specimens. Xia et al. demonstrated that quantitative T2-imaging with use of micromagnetic resonance imaging (with a 7.0-T magnet) can demonstrate the three histological zones of cartilage (tangential, transitional, and radial) on the basis of collagen fiber orientation
34 . This technique also has been used to demonstrate early changes in the superficial zone cartilage in a canine model of early osteoarthritis.
Knowledge of the human genome will have an enormous impact on all areas of medicine. The orthopaedic genome includes all of the genes that are involved in the formation, maintenance, and repair of the musculoskeletal system. Identification of the genes that direct and regulate the formation of bone, cartilage, and other tissues eventually may allow us to recapitulate the process that occurs during embryonic development. This information will provide a foundation for future approaches to tissue-engineering. As particular genes are identified, therapeutic targets will be defined. For example, the identification of parathyroid hormone-related protein (PTHrP) as an important regulator of bone formation has led to new treatments for osteoporosis. The sequencing of the human genome has also allowed the production of recombinant growth factors that are being used in orthopaedics. The best-known examples are probably BMP-2 and BMP-7, which are now available for certain clinical applications. The identification of the gene sequence for other cytokines will allow for the production of large quantities of these proteins and the potential for use in patients to augment healing and tissue formation in tissue-engineering applications.
New information about the human genome has, in turn, led to new understanding of the factors that regulate gene expression. It is now known that gene expression depends on the promoter region of the gene (DNA sequences that are upstream of the protein-coding sequence) and specific transcription factors. Transcription factors are activated by phosphorylation, which initiates an intracellular signaling cascade that eventually controls gene expression in that particular cell. Because intracellular signaling molecules are the final step in the pathway to the expression of a specific gene, precise control of gene expression will be possible with regulation of transcription factors. New research efforts in molecular orthopaedics will be directed toward the further identification of intracellular signaling molecules and pathways
35 . Manipulation of signaling pathways may be possible with use of specific agents, such as activators or inhibitors of kinases. This strategy could allow for the direct regulation and control of gene expression. For example, recent work has indicated that the inhibition of receptor-mediated retinoid signaling induces expression of chondroblast markers, including the transcription factor Sox9, which is a critical transcription factor in chondrocyte differentiation
36 . Identification of the retinoid signaling pathway provides the ability to modulate chondroblast differentiation.
As researchers have gained the ability to measure the expression of specific genes in an individual, it has become evident that different individuals can have widely varying responses to the same stimulus. Such differences in gene expression may be due to single base differences in the gene promoter. These single base differences are called single nucleotide polymorphisms. Thus, an individual's genetic background may influence his or her susceptibility to disease or his or her response to a certain stimulus. For example, the response to particulate wear debris likely depends on specific gene expression patterns. Determination of an individual's genetic background eventually will provide insight into the risk of certain diseases. Although such information will allow for dramatic improvements in medical care, there are obvious ethical concerns that will need to be addressed as well.
There are approximately 30,000 unique genes in the human genome. The sequencing of the human genome is really only a first step; the next part of the puzzle is to identify the subset of genes that is actually involved in a disease process. Gene arrays have been used to screen a large number of genes (as many as 10,000 or more) in a given cell population. Gene microarrays are now being used to define the molecular events in osteoarthritis. A recent study demonstrated that MMP-3 (stromelysin 1) is highly expressed in normal articular cartilage but is downregulated in late-stage osteoarthritis, whereas MMP-2 (gelatinase A) and MMP-13 (collagenase 3) are upregulated in late-stage osteoarthritis
37 . Although the role of these matrix-degrading genes has been established, gene array technology also results in the identification of many other genes, of unknown function, that are expressed at a high level in a given condition. Functional genomics is an important next step, which involves determination of the gene's role in normal and pathological processes. Functional genomics will be a rich area of investigation in the next several years.
Finally, the sequencing of the human genome will aid in continuing advances in the field of gene therapy. Since gene expression depends on the gene promoter, characterization of the promoters for specific genes will allow for more precise targeting in gene therapy applications. For example, the use of a promoter that is only expressed in a certain cell type would result in expression of the transgene only in that particular cell. Conversely, the use of a promoter that is expressed in a wide variety of cells would allow generalized expression of the gene. Moreover, the specificity of the promoter may also restrict expression to cells at certain stages of differentiation. Thus, the information gained from the human genome project may open new avenues for more precise gene therapy.
Any discussion of human gene research and the implications for treating, and even predicting, disease must include consideration of the ethical and social issues. In the future, it may be possible to determine an individual's predisposition to certain diseases. Should genetic screening become a routine part of the annual physical examination? There are obvious social implications to the knowledge that an individual is predisposed to a serious or fatal disease. Is the physician obligated to provide other family members with this information? There is the clear potential for such information to be used improperly by insurance companies and health-care providers. Patient safety issues will also be a major concern as new treatments (such as gene therapy approaches and new drugs) are developed and eventually pushed into clinical use by industry. These new treatments, although holding tremendous potential, are likely to be costly. Who will pay for such cutting-edge, costly treatments? These moral, ethical, social, and medical issues will need to be confronted, and orthopaedic surgeons will have to be able to participate in this discussion for the welfare of our patients and our specialty.
Lentivirus is a promising new vector for gene transfer. This vector provides high efficiency, long-term gene transfer, which may be especially valuable for the treatment of chronic diseases. Lentivirus-mediated gene transfer of the cDNA for human interleukin-1 receptor antagonist to the rat knee joint synovium was recently reported
38 . Interestingly, there was a positive biologic effect not only in the treated knee but also in the contralateral knee. A related development is the use of the TAT protein (a twelve-amino-acid protein sequence derived from the HIV genome) to transfer DNA or proteins into a cell (nonviral gene therapy). The TAT sequence facilitates the entry of a protein into the cell and thus may allow for the delivery of signaling proteins into cells. Recombinant proteins with an attached TAT sequence could be delivered to cells by local or systemic injection.
Gene transfer is being used as a complement to stem-cell therapy. Stem cells initially were isolated from bone marrow, but pluripotential cells are now being isolated from a variety of tissues. Cells capable of turning into chondrocytes, adipocytes, and osteoblasts have been isolated from blood, trabecular bone, fat, skeletal muscle, and synovium. Gene transfer to stem cells is being used to drive these cells toward a specific phenotype. It is also possible that more primitive cells, such as stem cells, are superior for tissue repair. Stem-cell research is likely to continue to be an active area of investigation.
This is an exciting time in orthopaedic research as advancements continue to be made in understanding the molecular basis of musculoskeletal diseases. Despite the increasingly technical complexity of contemporary research techniques, orthopaedic clinicians can and, in fact, should become involved in basic research. The very fact that research techniques are becoming more complex implies that such research will be carried out almost exclusively by basic scientists who may have little knowledge of the clinical problems and applications of their studies. This, coupled with the expanding potential to make important discoveries with these powerful techniques, points out the need for more clinician-scientists. An orthopaedist who understands the capabilities and limitations of the basic-science techniques and research findings will be a more effective collaborator. Similarly, the clinician-scientist can help the basic scientist to understand the capabilities and limitations of clinical data. The end result will be a more rapid application of basic-science information to clinical problems.
A very informative recent review by Brand et al., published in The Orthopaedic Forum of
The Journal, described the results of a survey of clinician-scientists with regard to their past and present funding
39 . The authors concluded that both the current number of clinician-scientists and the level of federal funding for musculoskeletal research are inadequate. That paper makes clear the need for more clinician-scientists in the United States. In recognition of this need, and to encourage orthopaedic clinicians to pursue basic research, the Orthopaedic Research and Education Foundation recently developed a Clinician Scientist Award. This award provides salary support of as much as $100,000 per year for up to three years to an orthopaedic surgeon who intends to spend 40% of his or her time in research. Application for this award requires a supporting letter documenting the applicants' intention to spend dedicated time in research. Applications for this award are due September 15 for funding in the next year. Similarly, the American Academy of Orthopaedic Surgeons recently formed a standing committee to identify and implement methods to increase the number of orthopaedic clinician-scientists.
Several upcoming meetings will provide timely updates on the latest findings, trends, and techniques in orthopaedic research for the clinician. The Seventh World Biomaterials Congress will be held in May 2004 in Sydney, Australia. The annual Grant Writing Workshop will be held in March 2004 at the offices of the American Academy of Orthopaedic Surgeons in Rosemont, Illinois. This meeting is aimed at assisting orthopaedic surgeons who are writing NIH RO-1 research grant proposals. Attendance at this meeting is limited to individuals who have written a draft NIH RO-1 research grant proposal. The annual meeting of the Orthopaedic Research Society will take place on March 7 through 10, 2004, just before the annual meeting of the American Academy of Orthopaedic Surgeons, and the Fourth Annual International Symposium on Tendons and Ligaments will be held on March 6, 2004, one day prior to the annual meeting of the Orthopaedic Research Society. Information about this meeting is available from Savio Woo, PhD, at the University of Pittsburgh. The American, Canadian, European, and Japanese Orthopaedic Research Societies will hold the Fifth Combined Meeting in Banff, Canada, from October 10 through 13, 2004.
The Orthopaedic Research Society offers two Traveling Fellowships per year. These fellowships provide funding for an orthopaedic researcher (a clinician or basic scientist) who is less than forty years old to visit one or several laboratories to learn new research techniques and to establish research collaborations. Further information about these Traveling Fellowships is available through the Orthopaedic Research Society.
In summary, there are numerous opportunities for orthopaedic clinicians, residents, and fellows to learn about (and to become involved in) the latest in orthopaedic research.
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