Extract
Innovation in orthopaedic basic science continues to abound. In this review, we highlight several notable studies published in the last twelve months with a focus on subject areas that were the topic of workshops at the 2009 annual meeting of the Orthopaedic Research Society (ORS) and that relate to the research of some of this year's Kappa Delta Award recipients. These papers and workshops reveal an exciting multidisciplinary approach to understanding mechanisms of tissue degeneration and injury—in particular, through the study of mechanobiology (physical effects on cells) and of aging. They also demonstrate important advances in the search for novel therapies to improve musculoskeletal tissue repair and regeneration through the use of stem cells, growth factors such as bone morphogenetic proteins (BMPs), or factors found in platelet-rich plasma as well as novel factors, particularly neurochemicals, molecules whose functions are primarily associated with the nervous system. A brisk pace in the clinical development of tissue-engineering scaffolds and refinement of total joint replacement materials is also evident.
Innovation in orthopaedic basic science continues to abound. In this review, we highlight several notable studies published in the last twelve months with a focus on subject areas that were the topic of workshops at the 2009 annual meeting of the Orthopaedic Research Society (ORS) and that relate to the research of some of this year's Kappa Delta Award recipients. These papers and workshops reveal an exciting multidisciplinary approach to understanding mechanisms of tissue degeneration and injury—in particular, through the study of mechanobiology (physical effects on cells) and of aging. They also demonstrate important advances in the search for novel therapies to improve musculoskeletal tissue repair and regeneration through the use of stem cells, growth factors such as bone morphogenetic proteins (BMPs), or factors found in platelet-rich plasma as well as novel factors, particularly neurochemicals, molecules whose functions are primarily associated with the nervous system. A brisk pace in the clinical development of tissue-engineering scaffolds and refinement of total joint replacement materials is also evident.
The importance of physical effects on tissue homeostasis, injury, and repair of connective tissues has long been recognized, and, as such, has been and continues to be the focus of much research. The 2009 annual meeting of the ORS featured two workshops relevant to this area. One workshop focused on the mechanosensory function of cilia in connective-tissue cells, whereas the other concerned the measurement of physical activity in patients. In addition, the Kappa Delta Young Investigator Award recognized the work of Stavros Thomopoulos (Washington University) on tendon mechanobiology. In this section, we review the workshops as well as several other relevant recent publications.
Primary Cilia as Mechanosensors
The subject of a recent review in the journal Cell1, cilia are currently a "hot topic" in many fields of research. First characterized on the basis of their motile function, cilia are now known to be present on many cell types, including nonmotile cells. Protruding like an "antenna" from the cell surface, cilia are a complex organelle with >800 proteins that participate in their functions, and mutations in some of these proteins result in human disease. New data implicate cilia in modulating the signaling of factors such as the hedgehog and wingless (Wnt) families of growth factors, and these data have made cilia particularly interesting in the study not only of skeletogenesis but also of development generally as well as of cancer and other areas. The presence and mechanosensory role of cilia in osteocytes, chondrocytes, and tendon cells was discussed in a workshop led by Cornelia A. Farnum (Cornell University) entitled "Primary Cilia: The Cell's Antenna for Sensing the External Environment."
Dr. Farnum's workshop began with a talk by Brad Yoder (University of Alabama), who presented an in-depth review of important advancements made in the last decade in terms of the role of the primary cilium in the pathogenesis of polycystic kidney disease as well as the potential role of abnormalities of primary cilia resulting in changes in whole-body metabolism leading to obesity, such as in Bardet-Biedl syndrome, which also has a skeletal phenotype of polydactyly. This presentation provided an overview of the kinds of experimental systems and mouse models, including transgenic models of Bardet-Biedl syndrome, that have been used in studies of the primary cilium in epithelial tissues. Thereafter, Christopher Jacobs (Columbia University) presented recent work from his laboratory related to the primary cilium of osteocytes that appear to act as sensors of fluid flow.
A recent study showed precocious osteoarthritis-like changes in mice with Bardet-Biedl syndrome, suggesting a role for primary cilia not only in bone but also in cartilage homeostasis. Kaushik et al.2 found that all three types of Bardet-Biedl syndrome mice that were examined (Bbs1M390R/M390R, Bbs2-/-, and Bbs6-/-) had significantly fewer ciliated chondrocytes in the joint cartilage, a lower proportion of flat chondrocytes in the superficial zone of the articular cartilage, thinner articular cartilage and subchondral bone, and less proteoglycan staining in the articular cartilage when compared with wild-type controls as assessed with histomorphometric analysis of the tibiofemoral joints. These findings are in agreement with those of Tony Poole (University of Otago, New Zealand), who was the second presenter at the ORS workshop concerning cilia. Dr. Poole presented a summary of his work exploring the hypothesis that the primary cilium of articular chondrocytes may act as a mechanosensor to the local biomechanical environment and may be significant in skeletal tissue cells in the establishment of cellular orientation and directed secretion of extracellular matrix components from the Golgi apparatus within cells. Recently, Dr. Poole's group investigated cilia in the chondrocytes of normal and osteoarthritic cartilage and reported an increase in ciliated chondrocytes in the clones of osteoarthritis-affected cartilage3. The additional observation that ciliated chondrocytes are more common in the columnar chondrocytes of the articular cartilage deep zone, close to the interface with subchondral bone, led the authors to propose that the increase in cilia may indicate the onset of chondrocyte maturation in these cells.
Little is known about the role of cilia in tendons, but a poster by Drs. Donnelly, Ascenzi, and Farnum (Cornell University and University of California at Los Angeles) demonstrated that the orientation in three-dimensional space of primary cilia in tenocytes is highly anisotropic and parallels the orientation of collagen fibers, supporting the hypothesis that these organelles are tenocyte mechanosensors as well. This poster was a winner of one of the ORS New Investigator Awards.
Physical Effects on Human Patients
Whereas the study of cilia pertains to responses to mechanical stimulation at the level of single cells and specific tissues, a workshop entitled "Physical Activity Assessment—Monitoring Patients During Activities of Daily Living," organized by Dieter Rosenbaum (Muenster, Germany), presented updates on new developments in our ability to assess the effects of physical stimulation at the whole-organism level in human patients. Gregory J. Welk (Iowa State University) presented information regarding new accelerometers that record not only the amount but also the patterns of movement and stressed how advancing computer technology has allowed the creation of smaller, more sensitive monitors with a higher data-storage capacity, all of which have contributed to making these devices increasingly more useful. Dr. Rosenbaum and Soren Brage (Cambridge, United Kingdom) followed with presentations that discussed the use of accelerometers in epidemiologic studies of outcomes in orthopaedic patients.
Tendon Mechanobiology
Several recent studies on the mechanobiology of tendon were notable this year, including one from Kappa Delta award-winner Dr. Thomopoulos. His group used a clinically relevant model of canine flexor tendon-to-bone injury and repair to show the beneficial effects of motion on the healing process4. In that study, the flexor digitorum profundus tendons of two groups of dogs were injured and repaired into bone tunnels in the distal phalanx. A 1-mm stainless steel bead was also sutured to the end of the tendon to mark its position in the bone tunnel. In the unloaded group the repaired tendon was cut proximally to prevent load transmission to the distal phalanx repair site, whereas in the loaded group the proximal tendon was left intact. The forelimbs of the animals in both groups were placed in a cast, and all animals were subjected to five minutes of passive motion daily. After twenty-one days, the tendons in the loaded group were found to have improved biomechanical properties, particularly in the physiologic subfailure range as well as improved range of motion at the distal interphalangeal joint. Loading was not found to affect bone mineral density or tendon gapping. It is unclear if the improved biomechanical and functional properties seen in the loaded group were due to increased load or increased motion. The tendon in the unloaded group was cut proximally, so it may have remained stationary in the tendon sheath during passive motion whereas the tendon in the loaded group was able to glide in the sheath. Previous studies of flexor tendon midsubstance healing demonstrated motion to be beneficial. This study provided valuable initial results; however, additional investigation is needed into the matrix synthesis at the gene and protein levels to identify the biologic mechanisms responsible for improved tendon-to-bone healing in the presence of controlled motion and loading.
Mechanical stimulation is important not only as a means to improve healing but also as a mechanism of injury. Studies show that overuse, underloading, and overloading all contribute to tendon injuries through different mechanisms. A new rat model of Achilles tendon overuse tendinopathy involving the use of a custom-designed uphill treadmill was reported by Glazebrook et al.5. The rat tendon displayed histologic features similar to those observed in human Achilles tendon disease, such as decreased collagen fiber organization and increased cellularity (endothelial cells and fibroblasts), and promises to be a valuable model for future investigations.
A group from the Cleveland Clinic studied the expression profile of the anabolic genes collagen I and decorin as well as the catabolic genes cathepsin K, MMP2, MMP3, and MMP13 in rat tail tendon fascicle explants over a forty-eight-hour time course of unloading in vitro. The anabolic genes collagen I and decorin, along with two of the catabolic genes cathepsin K and MMP2, demonstrated only small, fluctuating changes in expression level over forty-eight hours. Although collagen-I expression returned to normal levels by forty-eight hours, decorin remained slightly but significantly downregulated. However, when considered together, these anabolic genes did not appear to be significantly affected by mechanical unloading in culture. Similarly, expression of the catabolic genes cathepsin K and MMP2 stabilized to levels of the fresh explant by twenty-four hours and appeared to be insensitive to mechanical unloading in culture. On the other hand, the catabolic genes MMP3 and MMP13 were found to be highly sensitive to mechanical unloading as their expression was rapidly and dramatically increased compared with fresh explants and had not reached new homeostatic levels by the forty-eight-hour time point. Additional study of MMP3 and MMP13 over a longer time course is needed to identify the time needed for their expression levels to reach new homeostatic set points in culture. It is hoped that this future work will provide further insight into the mechanisms underlying the loss of tendon mechanical properties associated with mechanical unloading6.
Willett et al.7 recently studied changes in the thermal stability of collagen with mechanical overloading of a bovine tail tendon in vitro. It is theorized that disruption of the collagen lattice structure by permanent mechanical deformation enables greater conformational freedom of the thermally labile domain of individual collagen molecules, located at the gap regions of collagen fibers. This greater conformational freedom would allow for easier achievement of the thermal denaturation transition state, lowering the temperature of the onset of denaturation (Tonset) as well as the temperature of peak denaturation (Tpeak). However, the energy required to denature collagen, the enthalpy of denaturation (?H), is not expected to be altered as it is primarily dependent on collagen intramolecular bonding, which would not be affected by mechanical deformation.
In an attempt to substantiate the thermally labile domain theory, bovine tail tendons were ruptured under uniaxial tension at either 0.01 s-1 or 10.0 s-1. Differential scanning calorimetry was then used to study the thermal behavior of collagen from undamaged control regions of the tendon as well as damaged (but nonruptured) and ruptured regions of the tendon at both strain rates. It was found that tensile overload can significantly affect the thermal stability of collagen. Tonset was significantly decreased in damaged tendon regions compared with control regions at both strain rates. Tpeak was also significantly decreased in damaged tendon regions, with the exception of the nonruptured regions of tendons overloaded at the lower strain rate.
In addition to the findings supporting the thermally labile domain theory, this study also found an increase in the full width at half maximum of the endothermic peak, indicating an increase in the heterogeneity of thermal stabilities of collagen molecules within the tendons. The strain rate at which mechanical overload was applied to the tendon was found to affect Tonset, Tpeak, and full width at half maximum in the ruptured regions of the tendon. Regions ruptured at 0.01 s-1 were found to have decreased thermal stability and an increased heterogeneity of thermal stabilities. This finding may be explained by differences in failure mode at different strain rates. At lower strain rates, sliding of collagen molecules within the fiber may allow for a greater increase in gap regions compared with the ripping associated with rupture at higher strain rates. Additional study is needed to assess the effect of strain rate and to determine the biologic importance of the changes in thermal stability of collagen molecules. The findings of this study have lent substantial support to the conceptual model suggesting that damage to the lattice structure of collagen fibers leads to greater conformational freedom and subsequent decreased thermal stability. This information will be useful in future studies of collagenous tissues and will also provide insight into changes at the molecular level associated with mechanically induced collagen fiber damage7.
Aside from mechanical injury, aging is a major risk factor for the development of degenerative conditions of the musculoskeletal connective tissues. The workshop entitled "Orthopaedic Complications in Animal Models of Aging," organized by Christopher H. Evans (Harvard University), focused on the use of rabbit and mouse models to study the effects of aging in bones, articular cartilage, and intervertebral discs. These presentations highlighted the importance of oxidative damage, impaired DNA repair, and resultant cell senescence as key factors that contribute to the manifestations of musculoskeletal aging. The use of mouse models is also at the center of the work of Hiroshi Kawaguchi (Tokyo University), who was the recipient of the 2009 Kappa Delta Ann Doner Vaughan Award. Animal models were also discussed at the workshop entitled "Considerations for Orthopedic Product Development," during which the use of appropriate preclinical studies was a focus.
Animal Models of Musculoskeletal Aging
In the workshop on aging, Laura Niedernhofer (University of Pittsburgh) discussed several mouse strains whose progeria-like phenotypes point to the primary importance of accumulated DNA damage as a cause of aging. As explained in the recent review by Niedernhofer and Robbins8, several of the molecular mechanisms governing the response to the genotoxic stresses that accumulate in aging are known, and repression or overactivation of some of these—such as the anabolic factor insulin-like growth factor I (IGF-1), or the histone deacetylase SIRT1—can result in longevity in lower eukaryotes, confirming the importance of these molecules in determining lifespan. A similar strategy in mice, however, always appears to have a price, such as dwarfism, infertility, or glucose intolerance, for reasons that may relate to the complex functions of these molecules in "higher" animals. The studies by Joseph A. Buckwalter and James A. Martin (University of Iowa) were also presented at the ORS aging workshop; these studies focused on the importance of oxidative damage, telomere shortening, and cell senescence in chondrocytes as key factors in the development of osteoarthritis. Intervertebral disc degeneration in one of the progeria strains of mice described by Dr. Niedernhofer with impaired DNA repair (due to the decreased expression of the ERCC1-XPF gene) was presented by James D. Kang (University of Pittsburgh), underscoring the importance of DNA repair mechanisms in aging-related musculoskeletal diseases.
Dr. Kang also presented data indicating dramatic differences in tissue quality and gene expression between the surgical (needle annulotomy) and aging models of intervertebral disc degeneration in rabbits. The annulotomy model has become popular for the study of disc degeneration, and Elliott et al.9 recently reported that a needle-size-to-disc-height ratio of >40% determined the ultimate effects in the model by directly altering the mechanical properties of the disc through nucleus pulposus pressure deregulation or anulus fibrosus damage. Dr. Kang reviewed his own findings in the annulotomy model and described surprising significant differences with data from his recently published study on a cohort of aging rabbits10. His group found that in contrast to discs treated with annulotomy, those in aged rabbits showed increased levels of expression of BMP-2 and tissue inhibitor of metalloproteinases-1 (TIMP-1) and a decreased level of expression of transforming growth factor-ß (TGF-ß) in comparison with controls. The aging discs also were characterized by the presence of hypertrophic chondrocytes in the nucleus pulposus. On the basis of these observations, the authors suggested that mechanical injury and aging may affect intervertebral discs through different mechanisms.
A presentation by Regis J. O'Keefe, MD (University of Rochester Medical Center) centered on the role of cyclooxygenase-2 (COX-2) in the impaired healing of fractures in aged mice. His group recently reported the results of a study demonstrating that levels of COX-2, an important enzyme in the regulation of prostaglandin E2 (PGE2) synthesis, were significantly lower (by as much as 75%) in aged (fifty-two-week-old) mice as compared with young (seven to nine-week-old) mice five to seven days after experimental fracture, with associated decreases in chondrogenesis, callus vascularization, and bone formation in the older animals11. Normalization of the healing rate in aged mice after the administration of a prostaglandin receptor (EP4) agonist confirmed the primary role of COX-2/PGE2 in the aging-related delay in fracture-healing. Co-expression with receptor activator of NF?B ligand (RANKL) in the fracture callus suggested a role for COX-2 in controlling RANKL expression, a subject for future investigation.
Mouse Models of Bone and Cartilage Disorders
Mouse genetics have become a mainstay in the study of musculoskeletal biology and pathobiology, and this year's Kappa Delta Ann Doner Vaughan Award was given to Dr. Kawaguchi for his numerous contributions to the area. His paper was entitled "Mouse Genetic Studies on Molecular Mechanisms Underlying Bone and Cartilage Disorders," and his collaborators were Takashi Yamada, Toru Akune, and Naoshi Ogata. Exemplary of this work, Dr. Kawaguchi's group reported novel functions for the endosomal protein sorting nexin 19 and Krüppel-like factor-5 (KLF5)12 in chondrogenesis. Specifically, his group reported that mice that were haploinsufficient for the transcription factor KLF5 displayed delayed endochondral ossification during the perinatal period because of decreased expression of matrix metalloproteinase 9 (MMP9). Previous studies had implicated KLF5 in blood vessel growth.
The availability of innovative genetic and surgical animal models presents many exciting opportunities to uncover the mechanisms of musculoskeletal disease and injury healing, but this cannot be accomplished without appropriate methods for analysis of the relevant tissues. Two workshops at the 2008 meeting of the ORS focused on the use of imaging techniques to analyze bone and cartilage tissues.
Various techniques for evaluating bone and cartilage were presented by Drs. Carlson, Iwaniec, and Gronowicz at the workshop entitled "The Art of Bone and Cartilage Histology and Histomorphometry." The workshop emphasized histomorphometry as the "gold standard" because this method allows direct in situ analysis of bone and cartilage cells and their activities. In particular, the presentation featured the technique of dynamic histomorphometry, which is exquisitely sensitive because the fluorochromes that are used act as time markers that can limit the measurements to exclude bone that was formed prior to the treatment interval. Methodologic details recently reported by Dr. Iwaniec were presented13. Other specialized techniques were discussed, such as the use of frozen sections of undecalcified bone for visualizing multiple green fluorescent protein (GFP) transgenic constructs in mice, the use of terminal deoxynucleotidlytransferase-mediated deoxy-UTP nick end labeling (TUNEL) to detect apoptosis, and the use of bromodeoxyuridine (BrdU, a nucleotide analogue) to detect cell proliferation. Histomorphometric methods to quantify relevant parameters in cartilage tissue, particularly with the use of computer software (e.g., OsteoMeasure; OsteoMetrics, Decatur, Georgia) also were presented. The workshop stressed the value of histomorphometry as a critical tool for uncovering the information offered by the numerous transgenic and other animal models available for analysis.
Cartilage tissue analysis was also the focus of a workshop organized by Markus A. Wimmer, PhD, and Carol Muehleman, PhD (Rush University), entitled "Emerging Imaging Tools to Characterize Cartilage Damage." Vanguard technologies for the assessment of cartilage tissue morphology, biochemistry, and nanomechanics were described with substantial technical detail and examples of relevant data. Dr. Muehlman discussed diffraction-enhanced imaging, a novel radiographic technique that relies on the refraction of x-rays from different tissues. Knee joint images demonstrated the capacity for diffraction-enhanced techniques to image articular cartilage. Michel P. Laurent (Rush University) presented the use of scanning white light interferometry, which can be used to measure cartilage surface characteristics such as topography and roughness in live specimens with increased accuracy, efficiency, and speed in comparison with previously used contact-based stylus profilometry methods. In situ infrared imaging, which can also measure cartilage characteristics, was presented by Nancy Pleshko-Camacho, PhD (Exponent and Temple University). She reviewed three techniques for the spectroscopic imaging of articular cartilage, including (1) Fourier transform infrared imaging, which in conjunction with an array detector can be used to measure proteoglycan and collagen content as well as the collagen orientation of histologic and other thin samples, (2) in situ mid-infrared spectroscopy, which can be used to detect early degeneration on the surface of intact cartilage tissue, and (3) in situ near-infrared spectroscopy, which is the newest of the technologies. She explained that while near-infrared spectroscopy was appropriate for monitoring water, lipids, proteins, and sugars, because the overtone vibrations of near infrared are 100-fold weaker than those of mid-infrared, complex analysis is necessary to interpret the data. Alan J. Grodzinsky (Massachusetts Institute of Technology) discussed the use of atomic force microscopy-based techniques for the measurement of the nanomechanical behavior of cartilage tissue as well as that of cartilage matrix constituents with use of aggrecan as a case study. He showed how high-resolution force spectroscopy can be used to measure the nanoscale compressive forces between the atomic force microscopy probe tip and the aggrecan functionalized substrate as a function of tip-substrate separation distance and explained that by combining such height-normal load data with the force-distance data, an effective stress-strain curve can be calculated for these brush layers of aggrecan. As an example, he showed data indicating that fetal aggrecan was significantly stiffer than adult aggrecan, which is of great importance for correlating the nanomechanical properties of such matrix molecules with their molecular structure. Dr. Grodzinsky recently reported findings showing that aggrecan macromolecules can undergo Ca2+-mediated self-adhesion (because of both chemical and physical interactions) after static compression, even in the presence of electrostatic repulsion in physiologic-like solution conditions14. He postulated that such self-adhesion, and the macromolecular energy dissipation that results from it, could be an important factor contributing to the self-assembled architecture and integrity of the cartilage extracellular matrix.
Ultimately, the interest in mechanisms and models of disease and the need for methodologies to observe and record them stems from our desire to improve the healing and treatment of musculoskeletal injury, degeneration, and disease. From this point of view, the discovery of novel factors that control connective-tissue repair or regeneration is particularly exciting. The rest of this review focuses on discoveries that may be the basis of future therapies, such as the role of neurochemicals in musculoskeletal tissues, new insights into the nascent therapeutic use of stem cells, and new information regarding recently and well-developed clinical therapies such as the use of BMPs, tissue-engineering scaffolds, and total joint replacement.
At the Third New York Skeletal Biology and Medicine Conference, held in April 2009 at the Mt. Sinai School of Medicine, data were presented that revealed the surprising roles of neurochemicals in the biology and pathobiology of bone. In addition, recent studies have suggested important roles for neurochemicals in the degeneration of tendon and cartilage as well. Particularly interesting is that in most of these studies the effects of these neuropeptides and hormones—specifically, serotonin, oxytocin, and glutamate—are direct, occurring through the activation of receptors and/or the secretion of the neurochemical by the osteoblasts, osteoclasts, chondrocytes, and tenocytes themselves, and not secondary to central or peripheral neural effects.
Neurochemicals and Bone Volume
One of the most interesting studies in this area is from the group of Gerard Karsenty (Columbia University), who reported on the role of serotonin in the maintenance of normal bone mass15. Serotonin (5-hydroxytryptamine) is a neurotransmitter whose functions in regulating mood have been the basis of the widely used selective serotonin reuptake inhibitor class of antidepressants (such as Prozac [fluoxetine]). However, as much as 95% of the body's serotonin is produced in the gut by enterochromaffin cells and does not cross the blood-brain barrier, leaving the function of gut-derived serotonin obscure. Karsenty's group has shown that controlling bone mass is a major function of gut-derived serotonin.
The role of serotonin was identified through the study of mice lacking low-density lipoprotein receptor-related protein 5 (LRP5), a gene that controls bone density in mice and humans. The assumption had been that the role of LRP5 was to modulate the signaling of growth factors in the wingless (Wnt) family, for which LRP5 can act as a coreceptor. However, several lines of evidence suggested other mechanisms, including the observation that inhibition of the canonical Wnt signaling pathway in osteoblasts (by cell-specific deletion of ß-catenin) resulted in decreased bone resorption. This is in contrast to the LRP5-deficient mouse, which has decreased bone formation. Reexamining the findings from a previous microarray study in this mouse, the Karsenty group identified the gene encoding tryptophan hydroxylase 1 (Tph1), an enzyme whose action is the rate-limiting step in serotonin production outside the brain, as the most highly expressed in LRP5-deficient mice compared with wild-type controls. Consistent with this finding, LRP5-deficient mice were found to have a fourfold to fivefold increase in serum serotonin levels. Decreasing these levels (by dietary restriction of tryptophan) normalized bone mass in LRP5-deficient mice, confirming a role of serotonin in controlling bone mass. Additional in vitro and in vivo studies further defined the mechanism of action of serotonin by identifying Htr1b as the specific receptor through which serotonin affects osteoblasts and by showing that serotonin directly increases osteoblast proliferation through the transcription factor CREB. Perhaps the most surprising finding was that when mice with gut or bone-specific alterations of LRP5 were compared, gut LRP5 was observed to be more important for bone volume than osteoblast LRP5 was. This study also showed a protective effect of deleting Trp1 in the gut in the ovariectomy-induced model of osteoporosis, implicating gut Trp1 as a novel target in the treatment of postmenopausal osteoporosis.
The group of Alberta Zallone (University of Bari, Italy), in collaboration with the group of Mone Zaidi (Mount Sinai Medical Center, New York), reported on the anabolic effects of oxytocin, a neurohypophyseal hormone that controls social and feeding behavior, on bone16. In addition to its central action, oxytocin controls lactation. The investigators examined whether, on this basis, oxytocin also may control calcium homeostasis by regulating bone turnover. They found that mice lacking oxytocin or its receptor had decreased bone density. By comparing the effects of intraventricular and intraperitoneal injections of oxytocin, they found that—as with serotonin—the effects of oxytocin were not related to central nervous system functions but were peripheral and resulted in increased bone formation. Through in vitro studies, bone morphogenetic protein-2 upregulation in osteoblasts was identified as a mechanism by which oxytocin enhanced osteoblast differentiation. In osteoclasts, exogenous oxytocin resulted in direct upregulation of receptors of nuclear factor kappa-B (NF?B), but the pro-osteoclastogenic effects appeared to be modulated by the secretion of osteoprotegerin by maturing osteoblasts. As with serotonin, the apparent biologic distinction between central and bone-specific effects of oxytocin may make it attractive for the treatment of postmenopausal osteoporosis.
Glutamate in Tendon and Cartilage Degeneration
Another neurotransmitter, glutamate, is expressed both centrally and peripherally and is important for nociception in the context of injury or inflammation. Glutamate has previously been shown to be important for controlling bone turnover17. Recent studies also have indicated a role in tendon and cartilage tissue degeneration. For example, the expression of glutamate transporters (important for glutamate secretion) as well as increased joint fluid glutamate was recently reported in a rabbit anterior cruciate ligament transection model of osteoarthritis18. In addition, release of glutamate and expression of its receptors also was reported in chondrocytes by Piepoli et al.19. Those authors also found that while interleukin-1ß (IL-1ß, a major inflammatory cytokine in osteoarthritis) induced glutamate secretion by chondrocytes, blockade of the N-methyl D-aspartate receptor (NMDAR, a specific glutamate receptor) significantly diminished the upregulation of matrix metalloproteinases and cytokines induced by IL-1ß. The group of David Salter (University of Edinburgh, United Kingdom) previously showed the involvement of NMDAR-mediated hyperpolarization in chondrocyte mechanotransduction, and in 2008 that group reported a difference in the NMDAR complement in osteoarthritis-affected human chondrocytes20. Taken together, these reports suggest that glutamate may play an important role in the development of osteoarthritis.
Previous studies have established that excessive free glutamate can impact a variety of autocrine and paracrine functions important in the development of tendinosis, such as tenocyte proliferation and apoptosis, pain modulation, vascular function, and degenerative changes. The group of Sturen Forsgren (Umea University, Sweden) previously reported high intratendinous levels of glutamate in patients with tendinosis. In a further extension of that work, the investigators went on to investigate the origin of this intratendinous glutamate21. Vesicular glutamate transporters (VGluTs) are required for the transport of glutamate into secretory vesicles and therefore may be considered as indirect markers of potential sources of free glutamate release. Biopsies of both normal and tendinosis tendons (Achilles and patellar tendons) were examined immunohistochemically with use of antibodies against VGluTs. VGluT2 expression was detected in tenocytes (not nerves or blood vessels) and was significantly higher in tendinosis tendons as compared with normal tendons. In situ hybridization of VGluT2 demonstrated that mRNA was localized in a similar pattern as the protein, with marked expression by certain tenocytes, particularly those showing abnormal appearances. The study confirmed the presence of VGluT2 in human tenocytes and supported the hypothesis that free glutamate may be produced and released by tenocytes.
Nerve Growth Factor in Osteoarthritis and Ligament Repair
The group of Sturen Forsgren also recently reported on the expression of another type of neurochemical, the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) as well as their receptors p75, TrkA, and TrkB, in a mouse model of arthritis22. They found that expression of NGF, BDNF, and their receptors was increased in the inflamed synovium in structures described as "nerve fiber varicosities." Of interest, they also reported a significant decrease in NGF, p75, and TrkA in chondrocytes in the setting of arthritis, leading them to suggest a previously undiscovered role for these molecules in arthritis. While that study did not investigate any potential therapeutic role of replacing NGF in arthritis-affected chondrocytes, investigators from the University of Calgary reported on the reparative effects of exogenous NGF on rat medial collateral ligament healing23. NGF treatment resulted in an increase in nerve density and a decrease in expression of the angiogenesis inhibitor thrombospondin-2. Ligament vascularity was increased when measured with von Willebrand Factor (vWF) immunohistochemistry but not when measured in vivo with laser speckle perfusion imaging. NGF increased the failure load, ultimate tensile strength, and stiffness of the healing ligament. These results indicate that local application of NGF may augment ligament healing by promoting neovascularization and axonal proliferation, which can lead to improved mechanical properties of the developing scar.
The use of BMPs is no longer novel, with several products now available for clinical use for the treatment of nonunions and for spine fusion. However, with the application of novel techniques, studies of the basic biology of BMPs continue to reveal interesting new findings. Notably, this year, two studies on chondrogenesis and cartilage degeneration were based on microarray studies of micro-ribonucleic acids (miRNAs) to elucidate differences between BMP subtypes in transgenic mice, and stem cells and gene therapy were used for BMP-enhanced spine fusion.
Using miRNA to Study BMPs in Chondrogenesis and Cartilage
Microarray studies of miRNAs have been particularly useful for elucidating the functions of BMPs in chondrogenesis and cartilage degeneration. For example, Lin et al.24 used a comparative miRNA microarray approach to identify mi-R199a* as a downstream activator of BMP-2 signaling via Smad1. MicroRNAs are small noncoding RNAs that can control translation through complementary binding to target (coding) RNAs. Lin et al.24 reported that, during BMP-2-induced chondrogenesis of a mesenchymal stem cell line in micromass culture, mi-R199a* was dramatically upregulated after twenty-four hours. Following up on a computer-based prediction that Smad1 was the target for mi-R199a*, those investigators showed direct inhibition of Smad1 expression by 3'UTR binding. Excessive Smad1 reversed the mi-R199a*-induced inhibition of expression of early chondrocyte markers such as Sox9 and type-II collagen, confirming the interaction of mi-R199a* and Smad1.
In a noteworthy study, Iliopoulos et al.25 at the University of Thessaly in Greece reported on the combined use of miRNA microarray and proteomics to identify BMP-7 as part of a key gene network controlled by a specific miRNA in osteoarthritis. In that study, the authors used cartilage from normal (fracture) and osteoarthritis-affected patients to establish and compare miRNA gene signatures in the chondrocytes in each condition. Of interest, they identified several miRNAs, including mi-R22, that correlated with osteoarthritis severity (as determined on the basis of Mankin grading of the tissue) and with the patient's body mass index. They then performed reverse-phase protein microarray analysis of chondrocyte cell lysates and identified BMP-7, PPARA, and interleukin-1 ß (IL-1ß), among others, as proteins that also correlated with osteoarthritis severity and body mass index. Whereas many published studies have involved the use of gene expression microarrays to identify the target of miRNAs, those authors chose protein arrays because miRNAs control translation, not transcription, and target mRNA levels may or may not be significantly altered on the basis of miRNA activity against them. Additional in vitro testing revealed a role for mi-R22 in regulating levels of BMP-7 and PPARA, validating their miRNA/protein microarray approach. That study also identified several other miRNAs and other proteins (such as SOX11, FGF23, KLF6, WWOX, and GDF15) not previously associated with osteoarthritis to be differentially regulated in osteoarthritis as compared with normal cartilage, promising the elucidation of additional aspects of gene network regulation in osteoarthritis in the future. Previous studies have suggested that BMP-7 plays a specifically important role in articular cartilage, and the study by Iliopoulos et al.25 supported that contention.
Which Is the Best BMP for Bone Formation?
This question was at the center of a workshop run by Theodore Miclau III, MD (University of California at San Francisco) entitled "BMPs: Current Evidence and Future Solutions," which was held at the annual meeting of the ORS in 2009. The workshop focused on the use of BMPs for fracture-healing and bone repair and included a presentation by Emil H. Schemitsch, MD (St. Michael's Hospital, Canada) on a study showing the effectiveness of BMP-7 (also known as osteogenic protein-1, or OP-1) for fracture nonunions at his hospital. Also at this workshop, Thomas Einhorn, MD (Boston University School of Medicine) discussed the question "Are All BMPs the Same?" Following a detailed review of BMP biology, including receptor activation and downstream signaling, previously reported observations regarding the differences in the osteogenic capacity of several different BMPs were reviewed. He also presented data from his own laboratory that supported the primacy of BMP-2 for bone formation, including data from his 2006 study in mice with an osteoblast-specific deletion of BMP-2 showing that BMP-2 was not necessary for embryonic bone formation but was critical for postnatal fracture-healing26. A similar strategy was used to show that BMP-4 was not necessary for skeletogenesis or fracture-healing27. He also showed data from his 2007 study in which he demonstrated that while the inhibition of BMPs (by Noggin) increased or decreased the expression of BMPs depending on their subtype, only the inhibition of BMP-2 prevented the induction of bone formation by any BMP28. As mentioned by Dr. Einhorn, heterodimers of BMP-2 and BMP-7 have been shown to have greater osteogenic potency than either BMP-2 or BMP-7 homodimers. (BMPs are dimeric proteins, and expression of heterodimer proteins occurs in cells expressing genes of two different BMP subtypes.) In a recent study by Stuart Little and Mary Mullins (University of Pennsylvania), Drosophila embryos were used to show that BMP-2/7 heterodimers have specific effects based on their ability to heteromerize combinations of type-I BMP receptors29. Future studies in mammalian cells will show whether this mechanism is also important for bone formation and healing.
BMPs for Spine Fusion
In addition to fracture-healing, spine fusion continues to be an area for the clinical use of BMP. Currently, very large doses of BMPs are required for clinical effectiveness, and these agents rapidly consume large amounts of health-care dollars. In the early research on optimizing BMP delivery for orthopaedic applications, the vehicle with which it was delivered was examined, and the collagen sponge was determined to be the best option. Fu et al.30 reexamined the carrier for delivery of BMP-2 in a rabbit posterolateral fusion model and found that the use of an alginate matrix led to fusion rates of 83% with a dose that was fortyfold lower than had been reported in previous studies. Animals were divided into groups that received iliac bone autograft, alginate matrix and bone marrow-derived mesenchymal stem cells, BMP-2 (2.5 µg), alginate matrix and bone marrow-derived mesenchymal stem cells, or BMP-2 and alginate matrix alone and were fused at the L4/L5 level with use of the posterolateral intertransverse process model. Fusion as assessed with manual palpation was present in six of six autograft-treated animals, in five of six bone marrow-derived mesenchymal stem cell/BMP-2-treated animals, and in zero or one of the six animals in the other groups. Mechanical testing in torsion showed significantly increased maximum torque to failure in the autograft and bone marrow-derived mesenchymal stem cell/BMP-2 groups as compared with the alginate-bone marrow-derived mesenchymal stem cell group. Histologic analysis demonstrated that the alginate carrier degraded into tiny remnants and did not interfere with bone production, nor did it induce an inflammatory reaction. The authors concluded that with the alginate carrier and when codelivered with bone marrow-derived cells, dramatically lower doses of BMP-2 can be used to successfully induce bone formation and spine fusion in this comparative surgical model. Applied to clinical use, delivery of smaller BMP doses with use of an improved carrier may lead to smaller expenditures for these agents.
Gene therapy continues to be explored as an experimental method to improve BMP delivery. This technique typically involves genetically modifying cells to express genes of interest (in this case, BMP-2) with use of recombinant viral vectors. The cells may be those within the target tissue or ones that are genetically modified in cell culture and then implanted with use of surgical or injection-based techniques. Levicoff et al.31 compared the direct, in vivo administration of adenovirus or adeno-associated virus encoding BMP-2 into the intervertebral discs of rabbits and reported a greater prevalence of complications in the adenovirus group, including inflammatory changes in the disc, expression of the transgene by extradural cells and tissues, and the death of several animals, leading the investigators to conclude that adeno-associated virus is much safer than adenovirus for intradiscal gene delivery.
The group led by Jay Lieberman (University of Connecticut) continues to be particularly active in the area of gene-mediated BMP-2 delivery for spine fusion. Recently, that group reported on the increased effectiveness of a human immunodeficiency virus-derived recombinant lentiviral vector over a recombinant adenovirus vector for spine fusion and bone production in response to BMP-2 gene transfer in a rat model32. They used isogeneic bone marrow-derived mesenchymal stromal cells expressing BMP-2 to fuse the L4/L5 segment, and, although all of the animals had fusion, increased bone production and significantly more two-level fusions were found when the lentiviral vector was used. The exuberant bone production and unintended extra fused levels were attributed to the longer duration of expression obtained with lentiviral vectors, and the authors cautioned that lentiviral vector use will need to be optimized before its clinical application. In another study, the same group reported a 100% rate of successful spine fusion in an athymic rat model involving the use of a recombinant adenovirus to express BMP-2 in human adipose tissue-derived mesenchymal stem cells33. Furthermore, Miyazaki et al.34 reported a head-to-head comparison between human adipose tissue-derived mesenchymal stem cells and human bone marrow-derived mesenchymal stem cells in an athymic rat model for spine fusion and showed that adenoviral delivery of BMP-2 with use of either of these stromal cell sources produced spine fusion in all animals. Collectively, these studies suggest that, in the future, adult tissue-derived stem cells from various sources may be utilized for gene therapy but that additional work will be required to identify the ideal gene-transfer vector.
Stem cells are not simply vectors for growth-factor delivery. On the basis of their plasticity and enormous capacity for proliferation, stem cells continue to be attractive for the development of novel therapies for musculoskeletal injury repair. Recent studies have focused on three major areas, specifically, the mesenchymal differentiation of embryonic stem cells, the therapeutic use of mesenchymal stem cells, and the identification of new tissue-specific stem cells.
Mesenchymal Differentiation of Embryonic Stem Cells
Several investigators have reported on the mesenchymal, chondrocytic, and osteoblastic differentiation of embryonic stem cells35-42. Simple outgrowth of fibroblasts on plates coated with gelatin or Matrigel (BD Biosciences, San Jose, California) was used, although encapsulation in alginate without embryoid body formation was reported by one group to result in successful chondrogenesis and osteoblastogenesis40. One study by the group of Jennifer Elisseeff (Johns Hopkins University) evaluated the multilineage differentiation of embryonic stem cell-derived mesenchymal cells, including chondrogenesis, adipogenesis, and osteoblastogenesis. That study also demonstrated the successful repair of articular cartilage defects in rats by the implantation of pelleted embryonic stem cell-derived mesenchymal cells that had been expanded in the conditioned media of normal, differentiated chondrocytes38. In a similar study, Fecek et al.37 reported ectopic cartilage formation by embryonic stem cell-derived chondrogenic cells seeded onto polycaprolactone scaffolds.
Several groups have also reported the successful osteoblastogenic differentiation of embryonic stem cell-derived mesenchymal cells in standard osteogenic medium (containing dexamethasone and ß-glycerol phosphate)35-37,40,42. Jukes et al.41, however, reported that, in contrast to the above-mentioned studies on cartilage tissue formation, bone tissue formation was not demonstrated when osteogenic embryonic stem cell-derived mesenchymal cells were implanted into an ectopic site. Instead, successful bone formation was only achieved through a process of endochondral ossification where the embryonic stem cell-derived mesenchymal cells that had been subject to chondrocytic differentiation were implanted. Bone formation was most successful in association with the use of cells that had undergone chondrocytic differentiation for twenty-one days as compared with earlier time points, and these cells were capable of not only ectopic bone formation but also orthotopic bone formation in the context of a calvarial defect.
Resident Stem Cells in Adult Connective Tissues
While the improved control of embryonic stem cells remains an important area of research, the identification of tissue-specific stem cells is also of great interest as they may be simpler to obtain than embryonic stem cells and because their inherent existence and behavior may illuminate previously unknown aspects of the biology and pathobiology of the tissues in which they reside. The findings of recent studies have suggested that these local cells may be related to the tissue vasculature, although additional studies are necessary to characterize the association completely.
In the March 2009 issue of The Journal of Bone and Joint Surgery (American Volume), the group of Frederick S. Kaplan (University of Pennsylvania) reported on the identification of the osteoprogenitor cells that result in ectopic bone formation in fibrodysplasia ossificans progressiva43. Using a gene recombination study to trace the lineage of particular subpopulations of cells in mice, that group identified cells that were positive for the endothelium and the endothelial progenitor cell marker (and angiopoietin receptor) Tie-2 as the subpopulation that participated in the fibroproliferative, chondrogenic, and osteogenic stages of heterotopic ossification resulting from the injection of BMP-2 in a Matrigel carrier or in the context of cardiotoxin-induced muscle injury (which results in expression of BMP-4). Surprisingly, myogenic cells expressing MyoD and blood vessel smooth muscle cells expressing smooth muscle myosin heavy chain were not involved. That study did not rule out the possible contribution of muscle or perivascular-derived cells in fracture-healing or other bone-forming conditions, but it identified a new subpopulation of osteoprogenitors that are of potential value for therapies to improve or induce bone formation. From human rotator cuff tendons, Tempfer et al.44 isolated cells expressing CD133 (another marker of endothelial and hematopoietic stem cells) and also expressing Scleraxis (a marker of tendon precursor cells). Further characterization of these cells will be required to determine whether, as postulated by the investigators, they represent perivascular tendon precursor cells that may be activated for tendon repair.
The group of Nicolai Miosge (Georg August University, Germany) recently reported on the presence of migratory progenitor cells in the cartilage of patients with late-stage osteoarthritis45. These cells, which were isolated on the basis of their ability to migrate out of cultured tissue samples, were easily obtained from arthritic samples but not from normal cartilage, out of which cells did not readily migrate. When characterized, the migratory cells expressed markers of mesenchymal stem cells such as CD105 and CD73, among others, and were able to undergo chondrogenic, adipogenic, and osteoblastogenic differentiation in vitro. That group also showed that the cells could be transplanted onto cartilage explants into which they could migrate, leading the investigators to postulate that these cells may be useful for the repair of degenerated cartilage. Based on the presence of vascular invasion into the calcified cartilage in the arthritis samples described in their study, one also might speculate that these progenitors, like the ones that form ectopic bone or that are found in rotator cuffs, also may be of perivascular or vascular origin.
Bone Marrow-Derived Mesenchymal Stem Cells
While the use of bone marrow-derived mesenchymal stem cells is not novel in comparison with the use of embryonic or local tissue stem cells, research in bone marrow-derived mesenchymal stem cells has continued to evolve as they have progressed to clinical use. In one interesting study, Liu et al.46 reported improved proliferation and colony formation in bone marrow-derived mesenchymal stem cells that had been genetically modified to overexpress Nanog or Oct4, two of the genes that were previously used to induce an embryonic stem cell-like phenotype in adult dermal fibroblasts. In vitro differentiation studies showed that overexpression of either transcription factor improved chondrocytic differentiation but that only Oct4 improved adipogenesis whereas Nanog delayed it. Many differences were also detected in the Nanog and Oct4-expressing mesenchymal stem cells, indicating different targets for each transcription factor.
Whereas forced expression of key transcription factors is one way to control mesenchymal stem cell differentiation and/or behavior, mechanical loading presents a very different method that is likely critical for the differentiation of mechanosensitive musculoskeletal tissues and, in particular, the differentiation of tendon and ligament cells, a process that is currently ill defined. A recent study by the group of Rocky Tuan (National Institutes of Health) evaluated the expression of Scleraxis (a marker of tendon precursors) as well as that of Wnt4 and Wnt5a in mesenchymal stem cells stimulated with cyclic dynamic strain47. While mRNA levels for collagen were not significantly altered by strain, those of the matrix metalloproteinases were modulated, as was matrix deposition. Those studies are in agreement with previous tissue-engineering studies of chondrogenesis as well as previous developmental studies of tendinogenesis in highlighting the important role of mechanical stimulation in tissue formation.
Another cell-based approach that has recently become of great interest for bone and soft-tissue healing is platelet-rich plasma. Platelets contain a number of proteins, cytokines, and other bioactive factors that initiate and regulate basic aspects of wound-healing. The working definition of platelet-rich plasma is a platelet concentration of at least 1,000,000 platelets/µL in a 5-mL volume of plasma, and it is usually prepared from the blood of the patient undergoing treatment. It is believed that platelet-rich plasma enhances wound-healing through the delivery of various cytokines from the alpha granules contained in platelets, including transforming growth factor-ß (TGF-ß), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I, IGF-II), fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and endothelial cell growth factor (ECGF). These cytokines play important roles in chemotaxis, cell proliferation, cell differentiation, and angiogenesis.
Several recent studies have shown that platelet-rich plasma positively affects gene expression and matrix synthesis in tendon cells. Cell proliferation and total collagen production are increased in human tenocytes cultured in platelet-rich plasma, with slightly increased expression of matrix-degrading enzymes MMP 1 and MMP 348. A recent study showed that the injection of platelet-rich plasma into a patellar tendon injury site in a chimeric rat expressing green fluorescent protein in circulating cells and bone marrow cells resulted in increased recruitment of circulation-derived cells to the injury site, with concomitant increased collagen production49.
Murray and colleagues have performed a series of studies to evaluate the role of a collagen scaffold containing platelet-rich plasma in anterior cruciate ligament healing. One of the fundamental reasons for the ineffective healing response of the anterior cruciate ligament is the lack of a bridging scaffold joining the torn ends of the ligament. In a pig model, ligaments that had been treated with a collagen-platelet-rich plasma hydrogel demonstrated significant improvements in terms of maximum load, load at yield, and linear stiffness at four weeks in comparison with untreated repairs50. Platelet-rich plasma also may augment anterior cruciate ligament graft healing. The application of a collagen-platelet composite around a patellar tendon autograft anterior cruciate ligament reconstruction in a goat model resulted in improvements in knee laxity in comparison with that associated with grafts that had been treated with a collagen scaffold alone51. There were significant correlations between serum platelet concentration and anteroposterior laxity, maximum load, and graft stiffness.
In contrast to its use in tendon and ligament, the efficacy of platelet-rich plasma in the context of bone formation has not been uniform. For example, a recent study demonstrated no significant difference between platelet-rich plasma and platelet-poor plasma in terms of ectopic bone formation52. Another group recently reported no added effect of platelet-rich plasma in the setting of BMP-2-augmented spine fusions in a mouse model53. One possible explanation for the inconsistent findings was recently investigated by Han et al.54, who showed that platelet-rich plasma only improved the osteoinductive activity of demineralized bone matrix when used without thrombin activation. Another group, using a rat calvarium-healing model, presented evidence that the proportion of platelet-rich plasma to bone graft was an important factor in healing, with excess platelet-rich plasma being deleterious55. In light of these mixed experimental findings, a recent review demonstrating a lack of sufficient clinical studies to support the use of platelet-rich plasma for the treatment of delayed fracture-healing is not surprising56. Clearly, additional studies are required to discover whether the abundance of growth factors in platelet-rich plasma can be used effectively to augment bone-healing.
Even in its use for soft tissues, a number of important questions remain about the basic biologic mechanism or mechanisms of platelet-rich plasma. Additional information is required to examine the differential effects on acutely injured tendon as opposed to degenerative tendon, given the distinct differences in the underlying biology of these two disparate clinical conditions. The optimum time to inject platelet-rich plasma following acute soft-tissue injury must be determined because it is possible that the effects of cytokines may be very different at varying times after injury. Furthermore, the effect of serial injections should be explored. The factors that control the rate of cytokine release from different platelet-rich plasma preparations need additional study as this will help to determine the optimum timing for the injection of a specific platelet-rich plasma formulation.
While research on stem cells progresses, so does that on tissue-engineering scaffolds, some of which can support the growth of new tissue even without the implantation of cells. This year's Elizabeth Winston Lanier Kappa Delta Award went to Michael J. Yaszemski and coauthor Lichun Lu (Mayo Clinic College) for their work on scaffolds for skeletal defect repair. In this section, we also review several recent findings on the use of scaffolds for ligament and meniscus repair and regeneration.
Drs. Yaszemski and Lu's award-winning paper was entitled "Osteoinductive Injectable Degradable Polymeric Scaffolds for Osseous Defect Repair." On this subject, they recently published a report characterizing the porosity and mechanical properties of an injectable, biodegradable scaffold of poly (propylene fumarate) in which weak acids were used to form bubbles, resulting in the desired porosity57. In another study, Dr. Lu's laboratory combined biodegradable injectable poly (ethylene glycol) and dicarboxylic acids with methylmethacrylate into a cross-linking network58. They demonstrated cytocompatibility of the scaffold with osteoblasts and neural cells and an ability to control scaffod modulus by altering the method of manufacture. These are only two examples of several strategies the group has used to innovate bone tissue engineering. A comprehensive review of the topic, including the work by Dr. Yaszemski and others and coauthored by Dr. Yaszemski, appeared in a recent supplementary issue of The Journal59.
Small Intestine Submucosa for Ligament Repair
Porcine small intestine submucosa, when used as a bioscaffold, has unique characteristics that are useful to promote healing of ligament and tendon defects, including an ideally aligned collagen-I matrix with growth and chemotactic factors that are capable of stimulating cell migration into the healing site60. On the basis of previous studies showing that small intestine submucosa can enhance the healing of a 6-mm defect in the rabbit medial collateral ligament with improved collagen formation and mechanical properties at twelve and twenty-six weeks after an injury, the group of Savio Woo (University of Pittsburgh) investigated whether small intestine submucosa improves collagen morphology and organization as well as gene expression of fibrillogenesis-related factors at an earlier time point (six weeks)61. Healing of 6-mm medial collateral ligament defects over which a layer of small intestine submucosa had been sutured were compared with ligaments that had not been treated with small intestine submucosa and sham-operation ligaments in which no defect had been created. The application of the small intestine submucosa bioscaffold was shown to improve collagen fibrillogenesis, yielding larger-diameter fibrils that were more organized along the longitudinal axis of the ligament. These findings were more similar to those associated with the sham-operation ligaments than to those associated with the ligaments that had not been treated with small intestine submucosa, which had uniformly small collagen fibrils with no apparent organization. Previous studies have shown that higher levels of collagen V, decorin, biglycan, and lumican relative to collagen I are associated with poorer ligament mechanical properties and smaller, less organized collagen fibrils. In the study by Woo and colleagues61, gene expression of collagen V, biglycan, and lumican in the small intestine submucosa-treated ligaments were downregulated to levels that were significantly lower than those in the ligaments not treated with small intestine submucosa whereas collagen-I expression remained similar to that in the untreated group. The authors believed that the morphologic and molecular improvements at six weeks may have been responsible for the superior mechanical properties of the small intestine submucosa-treated ligaments at twelve and twenty-four weeks after injury.
Woo and colleagues also investigated the potential use of small intestine submucosa to prevent the donor-site morbidity that is commonly associated with bone-patellar tendon-bone autografts from the central patellar tendon that are used for anterior cruciate ligament reconstruction. In a rabbit model, a defect was created in the central patellar tendon and small intestine submucosa was placed at the base and over the anterior aspect of the defect. Previous studies have shown that the polarity of small intestine submucosa allows cellular infiltration only through its abluminal side62. Thus, it was hypothesized that in addition to promoting healing of the tendon defect, small intestine submucosa would prevent adhesion formation between the healing tendon and the infrapatellar fat pad. At twelve weeks postoperatively, the neopatellar tendon that had formed in the defect that had been treated with small intestine submucosa was found to have 68% higher cross-sectional area, 98% higher stiffness, and 113% higher ultimate load to failure. It also had denser and better organized collagen and improved vascularity. Additionally, the use of small intestine submucosa minimized adhesion formation. This indicated that the strategic use of small intestine submucosa with the luminal aspect oriented toward the infrapatellar fat pad limited cellular infiltration of the fat pad while promoting cellular infiltration and fibrillogenesis in the tendon defect. These findings are of particular clinical relevance because adhesions between the healing patellar tendon and the infrapatellar fat pad have been implicated in the anterior knee pain and patella baja complications that are frequently associated with harvesting of the central aspect of the patellar tendon63.
Scaffolds for Meniscal Repair
Non-cell-seeded scaffolds, which, like small intestine submucosa, are intended to encourage cell migration and tissue generation in vivo, have successfully transitioned to clinical use for the treatment of meniscal injury. For example, the Menaflex (ReGen Biologics, Hackensack, New Jersey) recently received approval from the United States Food and Drug Administration for use in the repair of partial meniscal defects. The implant is derived from devitalized purified and cross-linked bovine collagen and has been shown to support cellular ingrowth, to encourage tissue regeneration, and to improve postoperative clinical outcomes in comparison with the preoperative status and with the outcomes for partly meniscectomized knees at two years postoperatively64. Although the long-term function of the scaffold is as yet unclear, given the lack of clinically available options to treat partial meniscal defects, this treatment option likely will impact the management of patients who have local meniscal lesions.
Clinical data on the performance of a completely synthetic scaffold, based on a biodegradable porous aliphatic polyurethane (Actifit; Orteq BioEngineering, London, United Kingdom) are also starting to emerge. In preliminary data presented at the annual meeting of the European Society of Sports Traumatology Knee Surgery and Arthroscopy and at the International Society of Arthroscopy, Knee Surgery and Orthopaedic Sports Medicine (ISAKOS), vascular ingrowth into the scaffold in approximately 80% of patients65 and tissue ingrowth in as many as 86% of patients (Huysse et al.) were found at one year postoperatively. No evidence of cartilage damage was seen clinically, which was in contrast to the canine study of Welsing et al.66, who found that after twenty-four months of implantation the scaffold did not prevent degeneration of the articular cartilage of the tibial plateau relative to the meniscectomized controls. The discrepancy between the animal model and human studies may have been caused in part by the method used to attach the scaffold to the tibia in the dog model, which involved drilling holes through the tibial plateau, as compared with that used in the human, which involved suturing to the intact meniscal rim.
Total joint arthroplasty is a safe and effective procedure as an end-stage treatment for osteoarthritis. Total knee replacement, for example, has been associated with a survival rate of >97% at ten years; however, patients less than sixty-four years of age have a threefold higher rate of revision compared with their older counterparts67,68. In the case of hip replacements, the mean patient age has decreased from sixty-eight to sixty-five years over the past eight years (), raising concerns over implant longevity and the complications that occur in association with revision surgery. The dominant mode of failure of total hip replacements is aseptic loosening, which, in many cases, is caused by the reaction of bone to the presence of implant debris. In an attempt to increase implant longevity, bearing surfaces that minimize the volume of debris generated from the articular surface are being developed.
Ultra-high molecular weight polyethylene (hereafter called polyethylene), which has been the mainstay of arthroplasty, is a semicrystalline polymer consisting of amorphous areas interposed between highly organized crystalline regions. Its mechanical characteristics are directly attributable to its amorphous nature. For example, the ability of the material to resist crack propagation lies in the fact that while cracks can readily move through crystalline areas, the disorganized amorphous areas have the effect of arresting, or blunting, crack propagation. Efforts to improve the wear resistance of polyethylene have focused on increasing the degree of molecular cross-linking through the material, which has been shown to decrease wear rates considerably. The increased cross-linking occurs primarily in the amorphous phase, which has the undesirable consequence of reducing material fracture toughness. Therefore, although highly cross-linked polyethylene acetabular liners continue to demonstrate clinical wear rates and associated implant migration rates that are significantly lower than those associated with conventional polyethylene69,70, concerns remain about the clinical effect of its reduced fracture toughness. At the annual meeting of the ORS, Dr. Clare Rimnac and colleagues reported microscopically visible cracks in the rim of six of nine retrieved highly cross-linked polyethylene liners (Furmanski et al.). Of particular concern was the finding that one-half of all cracked liners had been retrieved after less than one month of implantation. The long-term consequences of these cracks are unclear, partly because it is unknown if the cracks will continue to propagate or if their growth rate will be arrested. By controlling the degree of cross-linking, the method of cross-linking, and the post-irradiation heat treatment conditions (annealing or remelting), the mechanical properties of the material can be controlled. This realization has led to second-generation highly cross-linked polyethylene71, which aims to improve wear resistance without sacrificing fracture toughness.
Changing the material with which the highly cross-linked polyethylene articulates has also been addressed in an effort to further improve wear characteristics. For example, OXINIUM (Smith and Nephew, Memphis, Tennessee), which is essentially a metal substrate with an oxidized (or ceramic) surface, has shown good short-term wear rates when used as a femoral head articulating against a cross-linked polyethylene liner72. However, substantial damage on the surface of retrieved dislocated OXINIUM femoral heads was recently identified73, and concern was raised about the potential of the damage to elicit substantially increased wear rates. In response to this concern, a wear study was conducted at Dr. Henrik Malchau's laboratory (Massachusetts General Hospital) to assess the effect of the femoral head damage on polyethylene wear. The investigators compared the wear performance of (1) retrieved damaged OXINIUM femoral heads on highly cross-linked polyethylene, (2) new OXINIUM femoral heads on highly cross-linked polyethylene, and (3) new OXINIUM femoral heads on conventional polyethylene. Over 2.5 million cycles, a small but significant increase in the wear of the retrieved damaged heads was found in comparison with that of the new femoral heads on highly cross-linked liners. However, the increase was relatively small compared with the wear of the non-cross-linked liners. The authors concluded that dislocation-related damage of oxidized zirconium heads would not lead to a catastrophic increase in wear.
Metal-on-metal articulations continue to gain in popularity, especially given the interest of young and active patients in receiving hip surface replacements. The long-term consequences of the release of metal ions continues to be monitored (Beaule et al.), and the indications for surface replacements now seem very much focused on young active male patients. The postoperative rate of implant-related osseous fracture remains around 2%74. Speculation about the cause of femoral neck fracture includes surgery-related disruption of the epiphyseal blood supply, notching of the femoral neck, varus alignment, and inadequate seating of the femoral component. In a statistically augmented finite-element-analysis approach, the laboratory of Dr. Donald Bartel recently reported that hip resurfacing increased the magnitude of the compressive strains in the inferior half of the peripheral neck by approximately 25% in the immediate postoperative condition75. They also found that bone modulus on the low side of the normal range and high head load contributed to large femoral neck strains. Their study suggested that the mechanism of femoral neck fracture was osseous damage accumulation, indicating that bone material quality should be assessed when screening patients for hip resurfacing and that rehabilitation protocols should avoid high-load activities.
Handouts for all ORS workshops can be downloaded at . A full list of Kappa Delta award winners can be found at . Transactions of abstracts from the ORS meeting can be searched and downloaded at . Transactions of abstracts from the ISAKOS meeting can be searched and downloaded at
Note: The authors thank the ORS workshop organizers who contributed executive summaries of their workshops to this manuscript (Thomas Einhorn, Cornelia Farnum, Gloria Gronowicz, and Dieter Rosenbaum).
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