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The Journal of Bone and Joint Surgery (American). 2005;87:2356-2365. doi:10.2106/JBJS.E.00569 © 2005 The Journal of Bone and Joint Surgery, Inc.
What's New in Orthopaedic Research1 The Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021. E-mail address for S.A. Rodeo: rodeos{at}hss.edu
Specialty Update has been developed in collaboration with the Council of
Musculoskeletal Specialty Societies (COMSS) of the American Academy of
Orthopaedic Surgeons.
Basic, translational, and clinical research in orthopaedics continues to be an important part of the mission of the American Academy of Orthopaedic Surgeons (AAOS). The AAOS Research Committee recently released a report entitled "Future Directions in Musculoskeletal Research." This report (available online at www.aaos.org/wordhtml/research/synthesis/panel_future_directions.pdf) is a detailed document that summarizes the findings of panels of experts in seventeen areas of musculoskeletal care. Each panel developed a report that described the scope of research, including its importance to public health as well as its clinical importance, recent advances in the field, and future directions for research. The research areas that were identified in the seventeen panel reports were sorted into twenty common themes ("Musculoskeletal Research Focus Areas"). The most common research themes identified by the panels were tissue-engineering, cell biology, genetics research (including biomarkers and gene therapy), biomechanics and biophysics, and outcomes research (including assessment tools and clinical trials). This summary will help to identify fruitful research topics for orthopaedic clinicians and scientists as well as students and scientists in training and also will identify areas in need of research support from funding agencies. This review will summarize the latest developments in several areas of orthopaedic research, including gene therapy, stem cells, tissue-engineering, and biomarkers. Specific topics that will be reviewed include fracture-healing, muscle injury, and osteoporosis and bone quality. We will also summarize information presented at joint symposia of the AAOS and the Orthopaedic Research Society (ORS) at their recent annual meetings.
The basic biology of bone-healing and methods to augment healing continues to be an active area of investigation. A combined ORS/AAOS symposium on biologic factors that can improve fracture-healing was presented at the recent meetings in Washington, DC. Several new developments will be reviewed here. Autologous blood concentrates have been approved for marketing by the United States Food and Drug Administration. This material contains growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß). Several commercial entities now market equipment that allows separation of platelets and plasma from red blood cells, concentration of platelets, and formation of a fibrin clot. This clot can then be used to augment bone-healing. At this time, there is very little clinical evidence to support its use. Exogenous cytokines continue to hold promise for the augmentation of fracture-healing. The angiogenic factor vascular endothelial growth factor (VEGF) was found to improve healing in a mouse femoral fracture model and a rabbit radial defect model1. Furthermore, inhibition of VEGF with neutralizing VEGF receptor resulted in impaired healing in a mouse femoral fracture model. Recombinant human bone morphogenetic proteins (rhBMPs), perhaps the most powerful osteoinductive factors known, continue to be investigated. Regulatory agency approval has been provided for the use of BMP-7 (also known as osteogenic protein-1) for the treatment of long-bone nonunions, and it also has been provided for the use of BMP-2 for spinal fusion. However, these applications require implantation of the protein on a collagen-based carrier material, thus necessitating direct exposure of the fracture site via open surgery. An important advance in this field has resulted from recent studies that have demonstrated the potential for percutaneous injection of BMP-2 to improve fracture-healing. Einhorn et al. created closed femoral fractures in rats and then percutaneously injected 80 µg of BMP-2 into the fracture site six hours later2. They found significant acceleration of fracture-healing according to both biomechanical and histologic criteria. At four weeks, the strength and stiffness of the rhBMP-2-treated fracture was equivalent to that of the contralateral, normal femur, whereas the untreated fractures and the fractures that had been treated with buffer only remained significantly weaker than the intact femur. Histologic analysis demonstrated extensive new-bone formation in the rhBMP-2-treated fractures. Recombinant human BMP-2 can be detected at the injection site for approximately seven days after injection with use of an aqueous buffer, whereas residence time can be extended to several weeks with use of a carrier. The residence time of rhBMP-2 was adequate to improve healing in the rapidly healing rat femoral fracture model; however, the next question to be examined was whether percutaneous injection could accelerate fracture-healing in slower-healing primate fractures. In a recently published study, Seeherman et al. found that rhBMP-2 delivered in an injectable calcium phosphate paste accelerated healing of fibular osteotomies in cynomolgus monkeys3. The rhBMP-2/calcium phosphate paste was injected under fluoroscopic guidance into the osteotomy site three hours after the osteotomy. The investigators found that the mean callus area, torsional stiffness, and maximum torque were significantly greater in the treated osteotomy sites as compared with the paired control osteotomy sites at ten weeks. Histologic analysis confirmed complete osseous bridging in the rhBMP-2/calcium phosphate paste group at ten weeks, whereas the paired control osteotomy sites were incompletely healed. The granular formation of the calcium phosphate carrier material was thought to play a positive role in the efficacy of injected rhBMP-2 in this model as the rh-BMP-2 binds to the granules and is gradually released over time as the carrier granules are resorbed. Furthermore, the granular nature of this carrier material causes the material to be dispersed over a wider area, allowing infiltration of blood vessels and cells. A new synthetic peptide drug, TP508 (Chrysalin), has been reported to enhance bone formation in preclinical models. TP508 is a twenty-three amino-acid peptide representing the receptor-binding domain of thrombin. At the site of bone injury, thrombin activates platelets and forms a fibrin clot. Thrombin is sequestered within the clot and is later released when the clot dissolves. Thrombin fragments released from the clot activate a cascade of growth factors and enzymes that initiate and regulate the healing process. TP508 mimics thrombin effects and appears to act by initiating the body's natural growth factor cascade at the site of injury. Sheller et al. reported significantly improved healing, as demonstrated with plain radiographs (p < 0.05), microtomography, and mechanical testing (p < 0.01), in association with the use of TP508 in biodegradable controlled-release poly(DL-lactic-co-glycolic acid) (PLGA) microspheres in critically-sized ulnar defects in rabbits4. In another study, TP508 improved consolidation of the regenerated bone following distraction osteogenesis in a rabbit model5. Quantitative computed tomography demonstrated significantly greater bone mineral density in the TP508-treated limbs as compared with controls (p < 0.05). Bone consolidation and remodeling were more advanced in the TP508-treated limbs compared with controls. The investigators who performed those studies are now planning studies designed to evaluate the effect of TP508 in articular cartilage regeneration. Although various demineralized bone matrix preparations have been available for clinical use for several years, there have been very few data comparing the various products on the market. Demineralized bone matrices are prepared by acid extraction of allograft bone, resulting in loss of most of the mineralized component but retention of collagen and noncollagenous proteins, including growth factors. Demineralized bone matrices have been used to improve spine fusion, to treat fracture nonunions, and to fill osteolytic lesions around total joint implants. A recently published study compared three different commercially available demineralized bone matrix preparations in a spine fusion model in athymic rats6. The three preparations that were tested included Grafton DBM Putty (Osteotech, Eatontown, New Jersey), DBX Putty (MTF [Musculoskeletal Transplant Foundation], available through Synthes, West Chester, Pennsylvania), and AlloMatrix Injectable Putty (Wright Medical Technology, Arlington, Tennessee). On the basis of radiographs, histological analysis, and manual testing of the retrieved spines, the animals treated with Grafton Putty had superior rates of fusion and more new-bone formation. Clinical studies are required to evaluate the efficacy of these products in patients.
Breakdown products of cartilage matrix molecules and chondrocyte metabolic products may serve as useful markers of the severity and progress of osteoarthritis. Most patients with arthritis are identified relatively late in the disease process, when a host of metabolic events have already occurred and the process is beyond the point at which pharmacological or surgical interventions can delay or reverse the process. The ability to detect the disease in its early stages may open the door for more effective interventions. The only widely available biomarkers that are in use at this time are bone markers (such as N-telopeptide) that are used for the evaluation and treatment of osteoporosis; despite a number of promising markers for articular cartilage metabolism, none have been validated to the point of being widely available for clinical use. A combined ORS/AAOS symposium entitled "Biomarkers of Osteoarthritis: Need, Challenges, and Potential Use" reviewed the current status of cartilage biomarkers. The utility of biomarkers will be for the early identification of patients who are at risk of rapidly progressive osteoarthritis, allowing for the selection of the most appropriate patients for pharmacologic or surgical therapy. Accurate biomarkers also will allow faster clinical trials of arthritis treatments as well as the evaluation of the response to such treatments. Biomarkers do not have to be only chemicals that can be measured in joint fluid (or blood or urine) but may also be indicators of cartilage matrix metabolism that can be detected with imaging modalities. For example, T2 relaxation time can be measured as an indicator of collagen organization in articular cartilage, whereas matrix proteoglycan content is reflected by measurement of the fixed negative-charge density of cartilage with use of delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC). Several substantial challenges to biomarker development exist at this time. Validation of currently available potential biomarkers is hindered by the absence of effective drugs for the treatment of osteoarthritis. Systemic (urine and serum) levels of a biomarker will have to be sensitive to events in a single arthritic joint. Furthermore, more information is required to understand how these chemicals may be metabolized in the body prior to their measurement in serum, urine, or even synovial fluid. Current biomarkers of interest include molecules that are released during matrix degradation and molecules that reflect repair processes. Aggrecan is a cartilage matrix molecule that is cleaved at several specific sites in arthritis. Antibodies that only recognize the cleavage fragments and identify the class of enzymes responsible have been produced. Similarly, antibodies that only recognize cleaved type-II collagen and not the intact molecule have been developed. As a marker for matrix turnover and repair, newly synthesized type-II collagen can be detected by propeptides that are released from procollagen II during collagen fiber formation. Further development of useful biomarkers will include consideration of the temporal specificity and process specificity of molecular markers. There is a temporal pattern of biomarker production and release. Current data suggest that aggrecan is released early in the arthritic process. The hyaluronan part of the molecule remains in the tissue and is released at a later point in the process. Fibromodulin and cartilage oligomeric matrix protein (COMP) are released at a later stage, and collagen cleavage occurs in the final phase. The term "process specificity" refers to enzymes and molecules that may be specific to a particular disease. This approach could help to distinguish inflammatory arthritis (such as rheumatoid arthritis) from osteoarthritis.
Studies from the laboratory of Huard et al. have contributed greatly to our understanding of the response of muscle to injury. Those investigators have studied the basic biology of muscle-healing in mouse models. The healing process in injured muscle involves degeneration and inflammation, regeneration, and eventual fibrosis. Studies of healing muscle have demonstrated that degeneration and inflammation occur in the early repair process. Muscle regeneration takes place in the first one to two weeks after the muscle injury, and then fibrosis begins during the second week after the injury. Scar tissue formation then gradually increases over time. Huard et al. evaluated the effect of several agents that can inhibit muscle fibrosis and can improve the number of regenerating myofibers. Those authors presented several studies at the recent meeting of the Orthopaedic Research Society. They reported that NS-398, a cyclooxygenase-2 (COX-2)-specific inhibitor, delayed the normal muscle-healing process by inhibiting muscle regeneration and promoting the formation of fibrous tissue in a gastrocnemius muscle laceration model in mice7. These findings were present at early time-points, but there were no differences between the groups by twenty-eight days after the injury, suggesting that the effect of the COX-2 inhibition is transient. In another study, those authors studied lacerated tibialis anterior muscles in mice and found that suramin (an antiparasitic and antitumor drug) inhibited fibrous tissue formation and improved muscle-healing after injury8. The mechanism of action of suramin appears to be inhibition of TGF-ß. There were more regenerating myofibers at the laceration site in suramin-injected muscles as compared with control muscles. Importantly, muscle function was improved in the treatment group, as demonstrated by significantly greater peak muscle contractile force as compared with that in controls that had been injected with saline solution only (p < 0.05). Another study from that group demonstrated that relaxin promoted improved muscle-healing with a greater number of regenerating myofibers in a mouse muscle-strain injury model. It was thought that this effect was due to relaxin-induced attenuation of TGF-ß at the muscle injury site. Because most clinical scenarios require the treatment of muscle fibrosis after it has already developed, Huard et al. also evaluated the effect of injection of matrix metalloproteinase-1 (MMP-1, collagenase) into an area of fibrosis that had formed following muscle injury in a mouse model9. The authors found that the MMP-1-treated limbs contained significantly more regenerating myofibers in the area of injury than did control limbs (p < 0.001). There was also significantly less fibrous tissue within the injury zone in the MMP-1-treated muscles as compared with controls (p < 0.05). These findings suggest the potential to ultimately treat muscle injury after scar tissue has formed. Another area of recent interest in muscle research is the effect of rotator cuff tendon detachment on the structure and function of the rotator cuff muscle. Muscle atrophy and fatty infiltration have been found to be important prognostic factors following rotator cuff repair. Gerber et al. studied the effect of infraspinatus tendon release and delayed repair in a sheep model to elucidate the changes in the muscle and to determine whether these changes could be reversed following tendon repair10. The authors found that retraction of the muscle-tendon unit following release was associated with profound structural and functional changes in the muscle, including muscle atrophy, fatty infiltration, and an increase in interstitial connective tissue. An important finding of that study was that there was fatty infiltration of the muscle rather than fatty degeneration. Muscle fibers did not appear to degenerate, suggesting the potential for recovery with appropriate stimulation. The amount of fatty infiltration was proportional to the degree of muscle retraction. Muscle retraction was associated with loss of elasticity of the muscle-tendon unit, which is consistent with clinical experience with the repair of long-standing, retracted rotator cuff tears. The structural changes in the muscle were not reversed after delayed repair of the tendon.
The Human Genome Project was very successful in sequencing the human genome. The next step is to identify the function of the molecules encoded in the human genome. Extensive research will be necessary to translate genome-based knowledge into improvements in medical diagnosis and treatment. A symposium at the recent annual meeting of the American Academy of Orthopaedic Surgeons entitled "Genomics: What Will Be Its Impact" explored ways in which information from the Human Genome Project may have profound effects on medical practice. The obvious application of the genome data is to develop new approaches to the diagnosis and treatment of many common diseases. We also may be able to develop genome-based approaches for the early detection of diseases such as osteoarthritis or osteoporosis and for the prediction of disease susceptibility. Furthermore, the genome data may allow for the development of a molecular taxonomy of various diseases. Understanding the genetic basis of disease ultimately may allow us to define diseases on the basis of the underlying molecular and genetic mechanism(s) rather than on the basis of symptoms. The genome information is also likely to have profound effects on drug development and how medications are used to treat various conditions. For example, pharmacologic therapy could be initiated for individuals who are genetically predisposed to a certain condition before that condition occurs. Perhaps disease-modifying agents for musculoskeletal conditions could be initiated prior to the onset of symptoms. Substantial improvements in drug therapy could be realized by individualized use of medication based on genetic variations in effects and side effects. Numerous important ethical questions also will need to be addressed as we consider the consequences of defining the genetic basis of disease susceptibility. How should this information be used in nonmedical settings? Policy guidelines will have to be developed to address this and other important questions. The social, legal, and ethical implications of genomics research will also require ongoing discussion and debate.
Clinical and experimental studies in the use of stem cells—genetically modified and otherwise—continue to be at the vanguard of evolving therapies in orthopaedics and other fields. The use of bone marrow-derived cells, or putative "mesenchymal stem cells" remains popular, in part, because they are easily harvested and manipulated. Of note, a clinical trial is being planned to evaluate the ability to regenerate meniscal tissue with use of allograft mesenchymal stem cells that are injected into the knee joint. Important new research has begun to elucidate aspects of the in vivo stem cell niche as well as the biology of stem cell differentiation. Embryonic stem cells also have been the focus of many new studies, and several laboratories have reported on the differentiation of these cells into osteoblasts, chondrocytes, and adipocytes. Key to the understanding of the genetic regulation of differentiation has been the study of transcription factors. Gene therapy remains integral to stem cell research, not only because of the need to control the behavior of stem cells genetically but also because stem cells promise to be excellent platforms for gene delivery. A major goal of clinical gene therapy remains the development of safe methods of permanent gene transfer, although for most musculoskeletal applications gene transfer needs to last only long enough to have the desired effect (for example, augmentation of meniscal healing). The in vitro isolation of single-cell clones with a high proliferative potential and ability to differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes, suggests that mesenchymal stem cells reside in the bone and bone marrow. However, little is known about the in vivo mesenchymal stem cells niche, such as where exactly in the bone they may reside, what the characteristics of that microenvironment may be, and also how the mesenchymal stem cells themselves may proliferate, migrate, and differentiate in vivo. Recent evidence suggests that interaction with hematopoietic stem cells is essential for the mesenchymal stem cells niche. A recent study by Mendes et al. demonstrated colocalization of mesenchymal stem cells with sites of hematopoiesis throughout embryonic development11. Furthermore, recent clinical studies have demonstrated improved grafting of hematopoietic stem cells when co-infused with mesenchymal stem cells. The group led by Elaine Fuchs at the Howard Hughes Medical Institute of Rockefeller University recently reported on a novel method of visualizing and tracking stem cells within skin in vivo12,13. Using genetically modified mice and genes expressed under skin-specific promoters, researchers in Fuchs' group were able preferentially to label stem cells in the skin with green fluorescent protein (GFP). Tracking these labeled cells has yielded a great deal of information regarding the characteristics and behavior of epidermal stem cells in vivo, including cell surface markers and specific stimuli that induce their proliferation and differentiation. It is possible that with the use of bone-specific and/or cartilage-specific promoters such as the osteocalcin or type-II collagen promoter, this type of strategy could be used to study cartilage and bone stem cells in vivo. The way in which mesenchymal stem cells differentiate into osteoblasts, chondrocytes, and other cell types also remains a central focus in the field of mesenchymal stem cell biology. Studies by the group led by Rocky Tuan at the Cartilage Biology and Orthopaedics Branch of the National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institutes of Health suggest that this differentiation is not linear and that transdifferentiation of one committed cell type to another can occur, at least in vitro. Song and Tuan reported that trabecular bone-derived mesenchymal stem cells that have differentiated into osteoblasts (as evidenced by their osteocalcin promoter-driven GFP expression) can be redifferentiated to express markers of either the chondrocyte or adipocyte phenotype14. Dedifferentiation seems to be an obligate middle process during this transdifferentiation. That group of investigators also examined the common and exclusive genes expressed during the differentiation of osteoblasts, chondrocytes, and adipocytes. The commonalities in these groups, along with the observation that some subsets of cells can differentiate into two but not all three of these cell types, suggest that, in addition to tripotent precursors, distinct osteoblast/adipocyte and osteoblast/chondrocyte bipotent precursors may exist. In the quest to understand how stem cell differentiation is genetically controlled, the study of transcription factors has become particularly important. Transcription factors regulate gene expression, orchestrating the turning on and off of a number of genes at once, usually by binding to a segment of DNA upstream of the coding region of specific genes (i.e., in a part of the genetic sequence that precedes the part that is actually transcribed and translated into a protein). Transcription factors often work together in discrete groups containing cofactors and other transcription factors. The specific groupings may affect whether a given factor will act to turn a target gene on or off. Developmental studies have elucidated several transcription factors that appear to act as "master regulators" of a specific cell type. Examples include the expression of peroxisome proliferator-activated receptor-gamma in adipocytes, Sox9 in chondrocytes, and Runx2 during osteogenesis. As these factors are expressed very early during the commitment of precursor cells, they are of interest not only in the discovery of mechanisms of differentiation, but also as markers of a particular cell type.
Studies on the Runx2 transcription factor (also known as core binding
factor 1 [Cbfa1] or polyoma enhancer binding protein 2A [Pebp2A]) exemplify
the importance of master transcriptional regulators as well as their complex
biology (as reviewed by
Komori15).
Haploinsufficiency of the Runx2 gene is the well-documented cause of
cleidocranial dysplasia, and, as expected from this phenotype, Runx2 is
critical for osteoblastic differentiation. Mice lacking Runx2 have
cartilaginous skeletons that do not ossify, pointing to the critical role of
Runx2 in chondrocyte maturation and subsequent ossification. Runx2 also
appears to be important in the early commitment of mesenchymal cells to
chondrocytes; however, as is the case in vitro, Runx2-deficient mesenchymal
stem cells tend to mature into adipocytes unless they are stimulated with
chondrogenic factors such as BMP-2. The Runx2 transcription factor is known to
form heterodimers with transcriptional coactivator core binding factor ß
(Cbfß/polyoma enhancer binding protein 2ß [Pebp2ß]), which
affect the affinity and specificity of their binding to target DNAs. It also
interacts with a number of other transcription factors, such as C/EBPß
and Transcription factor studies such as those mentioned above suggest that the coordinated regulation and subsequent expression of a number of genes may be possible by harnessing our understanding of the network of genes that are turned on and/or off by specific transcription factors that function as master regulators of a specific cell phenotype. In imagining the potential therapeutic applications of these studies, however, it is important to remember that much of our understanding of the transcriptional regulation of differentiation comes from observations made during limb development. Differentiation by postnatal stem cells is likely to follow a similar course and to depend on similar genes. However, whether and how developmental differentiation and postnatal stem cell differentiation may be similar or different are questions that have yet to be fully explored. Understanding the genetic basis of stem cell differentiation is a necessary prerequisite to controlling stem cells by means of gene therapy. The plasticity and proliferative potential of stem cells require that they be carefully controlled to prevent undesired transformation and/or overgrowth. Within their normal niche, stem cells rarely divide. Furthermore, they proliferate, migrate, and differentiate under a carefully coordinated sequence of local cues in their microenvironment. This knowledge underscores the importance of understanding the biology of the stem cell niche when considering the use of stem cells for clinical therapies. The combination of gene and stem-cell therapy also presents the possibility of effective permanent gene replacement, with stem cells being used as a platform for gene therapy. A modest number of genetically modified stem cells may be able to proliferate in vivo, potentially repopulating target tissues with a sufficient number of genetically "corrected" cells to have therapeutic impact. One of the most successful clinical gene-therapy trials to date involved the transfer of a corrective gene to hematopoietic stem cells for the treatment of X-linked severe combined immunodeficiency (XSCID). However, that trial also points to specific areas in which gene and stem-cell therapy need continued development. Whereas successful generation of genetically corrected, mature, functional T-cells and natural killer (NK) cells was detected in the blood of patients who were managed with hematopoietic stem cells modified by retrovirus-mediated gene transfer, at least two of those patients had development of T-cell leukemias. Whether the insertion of retrovirus genes into the host cell genome resulting in the overexpression of the growth-promoting LIM-only 2 (LMO2) genes actually caused the T-cell leukemia remains conjectural. However, that study and subsequent follow-up studies supported the concept that insertional mutagenesis is not as random or improbable as once imagined and that virus-mediated insertions, in fact, favor transcriptionally active sites, particularly when stem cells are "forced" to divide using cytokine stimulation16,17. Despite the various risk factors, viral gene delivery has dominated clinical trials primarily because of its in vivo efficiency when compared with nonviral methods. To improve the efficiency of nonviral gene transfer, methods are being developed in which viral and other elements are incorporated into synthetic carriers, such as polycation polymers of polyethylenimine, that bind DNA on the basis of charge interaction. Elements to enhance these synthetic carriers may include antibodies that bind receptors on the surface of target cells as well as viral and other proteins that may improve cell membrane translocation, intracellular tracking, and/or nuclear targeting. These modified or "hybrid" vectors have begun to be used in some clinical trials, particularly for the treatment of cancer. These hybrid vectors are designed to take advantage of the desirable aspects of viral gene delivery while eschewing the undesirable ones, including insertional mutagenesis16,17.
Regardless of whether genes are delivered by viral or other means,
site-specific integration is one way in which permanent gene transfer might be
made safer. Site-specific integration, of course, is now a routinely used
technology in the area of mouse genetics. For example, the Cre/lox system,
adopted from a prokaryotic gene for site-specific integration, has been widely
used to delete genes only in specific tissues. While prokaryotic genes cannot,
of course, be introduced into humans for the purpose of site-specific gene
transfer, studies have shown that endogenous sites that can accommodate
site-specific gene transfer do exist in the human genome. For example, the
bacteriophage integrase Therapeutic gene transfer may be particularly important for controlling the behavior of embryonic stem cells. Compared with mesenchymal stem cells, embryonic stem cells are more plastic and have a greater capacity for proliferation. As such, careful control of their phenotype as well as control of their growth will be critical to their safe use. Recent studies have demonstrated that genes such as BMPs can direct the differentiation of embryonic stem cells toward osteoblasts and chondrocytes18,19. Other studies are beginning to elucidate the signals, such as those of the Wnts, which can maintain cells in an undifferentiated or "stem" state. While embryonic stem cells hold great promise for future therapeutic applications, ethical considerations still abound. Further studies will be necessary to show what advantages embryonic stem cells may have over adult mesenchymal stem cells when considering orthopaedic applications.
Efforts to grow replacement tissues in vitro with mechanical, histological, and biochemical properties similar to those of the native tissue that they are intended to replace continues to be a focus of orthopaedic research. Variables during the preimplantation incubation period include the type of cell used (predifferentiated mesenchymal stem cells, undifferentiated mesenchymal stem cells, chondrocytes, osteoblasts), the material into which the cells are seeded (alginate, polycaprolactone, ceramics), the factors to which they are exposed (tumor-like growth factor, bone morphogenetic proteins), and the loading conditions to which they are subjected (no loading, hydro-dynamic loading via bioreactors). Recently, these parameters have been varied in an attempt to engineer bilayered composite tissues. For example, Tanaka et al. described a bioceramic ß-tricalcium phosphate block over-laid with chondrocytes seeded in a collagen gel for the repair of osteochondral defects20. Even though ß-tricalcium has been used to support bone-tissue formation since the 1970s, its use in the engineering of a composite tissue is novel. Thirty-week follow-up in a rabbit trochlear osteochondral defect model demonstrated resorption of the osseous substitute in tandem with osseous growth and synthesis of a cartilaginous-like matrix. However, the superficial zone was comprised mainly of fibrous tissue and the newly formed cartilaginous tissue did not integrate with the surrounding cartilage. Schek et al. also described a "biphasic scaffold" for repairing osteochondral defects21. The osseous layer consisted of porous hydroxyapatite seeded with fibroblasts that had been infected with a BMP-7-expressing adenovirus. The cartilaginous layer consisted of chondrocytes seeded into a porous poly-L-lactic acid polymer. The two layers were separated by a thin film of polyglycolic acid. Implantation into a subcutaneous mouse model after four weeks revealed the concurrent formation of cartilaginous material and bone, with a mineralized tissue interface in between. The newly formed cartilage-like material stained only slightly for proteoglycans, and osteogenesis occurred in the polymeric phase, possibly as a result of migration of the transfected cells. In all of these studies, and in many others, the engineered tissue is predominantly characterized with use of histological or biochemical techniques—for example, the proteoglycan content of the tissue-engineered cartilage is frequently quantified. The swelling pressure generated in the cartilage matrix by proteoglycans can resist compressive loads. Thus, although a measure of proteoglycan content can help us to infer the mechanical properties of the tissue, it is the interaction of the proteoglycans with the collagen network and the permeability of the tissue that truly determine the tissue's response to load. The only way to truly assess the functional performance of the tissue is to measure it mechanically. Rarely are the mechanical properties of the engineered constructs characterized, which makes understanding their true functional performance impossible. Many research groups are using mesenchymal stem cells for tissue-engineering applications. Alhadlaq et al. used a single population of mesenchymal stem cells in combination with a novel mold design and polymers to engineer a femoral condyle22. Bone marrow-derived mesenchymal stem cells were predifferentiated down an osteogenic or chondrogenic lineage and were encapsulated in stratified polyethylene glycol-based hydrogel layers, which were shaped to have a geometry similar to that of a femoral condyle. The gels were photopolymerized and implanted subcutaneously in a rat model. Both in vivo and in vitro data showed the expression of chondrogenic and osteogenic markers within these layers as well as the respective progression of tissue formation. That study suggested that a single population of mesenchymal stem cells implanted in vivo into a suitable scaffold can lead to the in vivo production of a cartilage/bone composite. Tissues for the replacement of the complete intervertebral disc, consisting of the anulus fibrosus and nucleus pulposus, also have been formed and grown in culture as composite tissues. In the study by Mizuno et al., cells isolated from the anulus fibrosus were seeded into a porous mesh of polyglycolic acid/polylactic acid (PGA/PLA) and cells isolated from the nucleus pulposus were suspended in alginate and injected into the center of the PGA/PLA mesh23. The constructs were implanted into a subcutaneous mouse model for periods of as long as twelve weeks. Histological and biochemical analyses revealed collagen and proteoglycan quantities similar to those of native discs. However, the newly formed tissue in the anulus fibrosus was not directionally organized as was the case with native tissue; this finding was possibly attributable to the non-physiological loading to which the constructs were subjected in the subcutaneous model. Furthermore, the nucleus pulposus had low levels of collagen (<10% of that of the native tissue). Again, although the mechanical characteristics of the newly formed tissue were not characterized, that study nonetheless represented a novel approach to the composite engineering of a replacement disc.
It has been estimated that ten million Americans over the age of fifty years have osteoporosis, while another thirty-four million are at risk for development of the disease24. To more effectively treat osteoporosis and to help to identify the disease at an earlier stage, it is vital that we understand more about the factors that contribute to overall bone properties. New technologies that more closely elucidate the molecular and crystalline structure of mineralized tissues are being developed; two such techniques are Fourier transform infrared spectroscopy (FTIR) and 31P solid-state nuclear magnetic resonance imaging. FTIR can be used to measure the absorption of various infrared light wave-lengths, and the absorption bands can identify specific molecular components and structures. Analysis of calcified tissues with use of FTIR allows one to measure the relative amount of mineral and the arrangement of apatite and organic matrix. 31P solid-state nuclear magnetic resonance imaging can be used to quantify the mass of hydroxyapatite in the tissue. It is envisaged that these combined tools will lead to a more complete indicator of the risk of bone fracture than is provided by bone mineral density measurements alone. Efforts also are being directed toward the development of bone-tissue substitutes. Xu et al. used calcium phosphate cement that hardens in situ to form solid hydroxyapatite25. The strength and toughness of calcium phosphate cement was increased with use of chitosan and mesh reinforcement, and macropores were created for bone ingrowth. With excellent biocompatibility combined with mechanical properties that can be tailored to mimic that of bone, the material is a suitable candidate for orthopaedic applications requiring bone reconstruction. Bioresponsive materials respond and remodel in response to biological signals from newly forming tissue. Their particular advantage for use as scaffolds in tissue-engineering applications is that they allow for the generation of new tissue in tandem with scaffold resorption. Wang et al. expanded on this concept by linking a phosphoester to a polyethylene glycol-based bioresponsive material in order to enhance in situ mineralization26. Mesenchymal stem cells that had been incubated in osteogenic medium were encapsulated and incubated for six weeks. Expression of osteogenic-relevant markers (osteonectin and alkaline phosphatase) was found; the constructs became calcified and appeared to be differentiating toward osteogenesis. While preliminary, that study emphasized the capabilities of further enhancing bioresponsive materials for the purposes of bone-tissue engineering.
In response to the challenges of regulating the clinical introduction of tissue-engineered-cell-based constructs, an advisory committee known as the Cellular, Tissue, and Gene Therapies Advisory Committee was convened by the United States Food and Drug Administration in March 2005. In the first of many such meetings, the advisory committee (formerly known as the Biological Response Modifiers Advisory Committee) started to discuss specific concerns from industry and academia about ways to ensure the safe and effective clinical use of cellular, tissue, gene-transfer, and other biological products. Transcripts from this meeting are available online at www.fda.gov/cber/advisory/ctgt/ctgtmain.htm. An additional challenge is to accurately assess the progressive growth and integration of the implanted tissue over time. While postoperative biopsies provide useful information, surgically removing tissue from a repair site is far from ideal. Enhanced imaging techniques such as phase-contrast magnetic resonance imaging have enabled the structure and content of repair tissue to be examined in a noninvasive manner. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) is a relatively new imaging technique that has been used to estimate joint cartilage glycosaminoglycan content. A hydrophilic negatively charged contrast agent, Gd-DTPA(2-), is injected into the joint and distributes in inverse relation to the concentration of negatively charged proteoglycans. A magnetic resonance imaging scan, followed by measurement of T1 relaxation time, allows the Gd-DTPA(2-) concentration in the tissue to be computed. Short T1 relaxation times in dGEMRIC correlate with low proteoglycan content, and prolonged T2 relaxation times correlate with decreased collagen organization27. The ability to add a tracking agent to tissue-engineered constructs for the purposes of tracking their performance with use of imaging is a powerful capability. For example, Bull et al. described novel self-assembled peptide amphiphile nanofibers that were conjugated to contrast agents to enable tracking of gel scaffolds with use of magnetic resonance imaging28.
The development of so-called "combination products" that combine traditionally implanted materials (such as cobalt alloys, titanium, or stainless steel) with biological factors that are added to enhance osseous fixation or bacterial resistance has become a more active area of orthopaedic research. For example, Lu et al. developed titanium-alloy discs coated with a biomimetically coprecipitated layer of calcium phosphate and BMP-229. After five weeks of implantation in a rat model, the coatings induced bone formation at an ectopic site and sustained this activity for a considerable period of time. Nablo et al. described nitric oxide-releasing gel films as antibacterial coatings for orthopaedic implants30. The gels consisted of 40% N-aminohexyl-N-aminopropyltrimethoxysilane and 60% isobutyltrimethoxysilane. The coated surfaces had statistically significant less bacterial adhesion of Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis as compared with uncoated steel and steel coated with the gel alone. Alumina nanofibers have also been used to enhance implant fixation31, with the hypothesis that the nanostructure is more suited to controlling the adhesion of nanoscaled biological structures (such as proteins and ligands) as compared with the µm-scaled structure of the metallic alloys traditionally used. Various crystalline-structured nanofiber pieces of alumina were manufactured, and, after fourteen days of culture with osteoblasts, alkaline phosphatase activity and calcium deposition were increased as compared with uncoated surfaces. The results were highly dependent on crystalline structure.
Highly crosslinked ultra-high molecular weight polyethylene (hereafter called crosslinked polyethylene) was approved for clinical use in the United States in 2000. Digas et al. used radiostereometric analysis in a prospective, randomized clinical study on patients who received either highly crosslinked or conventional polyethylene liners32. At two years, a 62% reduction in femoral head penetration was found when crosslinked liners were compared with conventional liners. Researchers are already developing a second generation of crosslinked polyethylene in an effort to improve baseline material properties, such as fracture toughness and strength. The main difference between the first and second generation of materials is the method of manufacture. The first generation of crosslinked polyethylene is irradiated to produce molecular cross-linking and then typically is heat-treated or annealed to eliminate free radicals, whereas the new generation of materials is manufactured with use of repeated bursts of radiation, each of which is followed by successive heat treatment. In early 2005, Biomet received marketing approval from the United States Food and Drug Administration for its second-generation highly crosslinked polyethylene, ArComXL. Since 2003, the following designs have secured Food and Drug Administration approval for use in the United States: Reflection hip (Smith and Nephew, Memphis, Tennessee), Transcend Acetabular System (Wright Medical Technology), Trident Ceramic Hip System (Stryker Orthopaedics, Mahwah, New Jersey), and the Keramos Acetabular System (Encore Medical, Austin, Texas). As the United States' experience with these implants continues to grow, the prevalence of clinical fracture appears low. However, it remains unclear as to how resistant these devices are to repeated cyclic impingement. Zirconium oxide, or Zirconia, has been used since the 1980s in the ceramic-polyethylene bearing combination of total hip replacements. Zirconia can exist in three different phases, or states of atomic bonding, depending on its temperature. The strongest and toughest form is called the tetragonal phase, which is naturally stable at temperatures of >1000°C. This phase can be artificially stabilized through the addition of yttria (5% by weight). However, if the phase becomes unstable, the material (or parts of the material) reverts to a weaker monoclinic phase. It was the uncontrollable transformation of Zirconia in vivo from its stronger to its weaker phase that led to the 2002 recall of ceramic heads manufactured by Saint Gobain Advanced Ceramics Demarquest (Monreuil, France). Efforts by Deville et al. to identify tools that can be used to determine the tendency of yttria-stabilized Zirconia to undergo phase transformations will ideally lead to preclinical tests for the screening of Zirconia heads under simulated in vivo conditions33. Oxinium, the Smith and Nephew trade name for oxidized zirconium, is a metallic alloy, the surface of which has been oxidized to form a ceramic substrate. The concept of the material is that the ceramic surface layer will provide improved wear resistance without the brittleness normally associated with ceramics. Despite showing a 42% reduction in wear when compared with cobalt-chromium alloys during in vitro knee-simulator tests34, the cementless versions of the Oxinium knee implants (Genesis II and Profix II knee replacement systems) were recalled in 2003. It appears that a lack of osseous growth into the implant followed by implant loosening led to the recall.
The SB Charité III total disc replacement design (DePuy Spine/Johnson and Johnson, Raynham, Massachusetts) was approved for clinical use in the United States in 2004. The SB Charité III consists of two end plates that are made of cast cobalt-chromium-molybdenum alloy. Each end plate has teeth that facilitate fixation into the adjacent vertebral bodies. The end plates are separated by a spacer made from ultra-high molecular weight polyethylene. Earlier concerns about osteolysis caused by the presence of wear debris in the spine appear to be unfounded, although it remains unclear where the debris will migrate and what effect it will have at other anatomical locations. The ProDisc system (Spine Solutions/Synthes) also consists of two cobalt-chromium-alloy end plates and a polyethylene inlay. ProDisc is the only artificial disc undergoing Food and Drug Administration trials for the treatment of multiple-level lumbar disc disease. Other total disc replacement designs undergoing Food and Drug Administration review include the Maverick metal-on-metal articulation system (Medtronic Sofamor Danek, Minneapolis, Minnesota), the FlexiCore system (SpineCore/Stryker Spine, Allendale, New Jersey), and the Bryan Cervical Disc System (Spinal Dynamics/Medtronic Sofamor Danek, Mercer Island, Washington). For earlier stage spine problems in which the surrounding soft tissues are structurally intact, implants that do not replace the entire disc but that replace the nucleus pulposus alone are being developed and tested preclinically35. Unlike the total disc replacements, implant stability relies heavily on the surrounding soft tissues and necessitates minimally disruptive surgical techniques to insert the implant and to ensure continuing functionality of the ligamentous structures.
The ORS is the premier organization in our field for dissemination of research findings. The annual meeting is held each year just prior to the AAOS Annual Meeting. The fifty-second annual meeting of the ORS will be held on March 5 through 8, 2006, in New Orleans, Louisiana. The ORS web site contains a listing of members who are willing to serve as mentors for young scientists. These individuals have expressed willingness to serve in various capacities, such as providing brief telephone consultations, performing confidential review of manuscripts and/or grants, or hosting a young scientist for a short period of time to teach a new research technique. The ORS also sponsors a Career Development Fellowship that provides as much as $7500 of funding for an individual to spend time at another institution. The goals of this program are to provide education for the recipient, to foster interdisciplinary collaborations, and to promote the interchange of ideas. The ORS also sponsors various research awards, and information is available on the ORS web site. The AAOS web site is another valuable source of up-to-date information on upcoming AAOS-sponsored symposia, scientific meetings, and reports from AAOS research committees. The web site also lists the various committees under the Council on Research. Individuals interested in research are encouraged to consult this web site and to consider applying for committee membership. Information is available on research awards, research funding, and the American Academy of Orthopaedic Surgeons/Orthopaedic Research and Education Foundation (AAOS/OREF) fellowship. Another important way for clinicians to become involved in research is to serve as reviewers on National Institutes of Health study sections. There is currently a critical need for orthopaedic surgeons to serve on review panels. In summary, orthopaedic research continues to advance at a rapid pace as new techniques are applied to musculoskeletal tissues. The discovery of biologic solutions to important problems such as fracture-healing, soft-tissue repair, osteoporosis, and osteoarthritis continues to be an important research focus. At the same time, research in biomaterials and biomechanics is critical to advances in current areas such as tissue-engineering and cytokine delivery.
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Introduction
Accelerating the Fracture Repair...
and also ETS1, as well as with BMP signaling molecules Smad1 and
Smad5, to upregulate the expression of osteoblast-related genes such as
osteocalcin and osteopontin. 
31 has been used successfully to integrate the
human coagulation factor-IX gene into "hot spot" pseudo-sites
(called mpsA and mpsL1) in mice with resultant increases in the circulating
factor-IX level. Current strategies can only insert genes into such sites at
low frequency. However, continued research in this area holds promise for
safe, permanent gene transfer in the
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