Finding novel interventions to treat patients in the early stages of disease through improved understanding of the molecular mechanisms of musculoskeletal disorders has been a focus of orthopaedic research. Over the past twelve months, important advances have been made in understanding the genetic and molecular basis of osteoporosis, osteogenesis imperfecta, and degenerative disc disease; furthermore, molecular pathways that could lead to regenerative therapies have been identified. Although it may take some time before these findings translate to clinical innovation, new and improved implants for end-stage joint degeneration are continually being optimized and analyzed. When taken together, it is clear that a continuum of care for patients with musculoskeletal problems is beginning to emerge. To highlight this continuum, here we present some of the more important findings across the spectrum of understanding and treatment of musculoskeletal disease. In particular, we summarize specific work as presented at the recent meetings of the American Academy of Orthopaedic Surgeons (AAOS), the Orthopaedic Research Society (ORS), and the American Society for Bone and Mineral Research as well as at the International Meeting on Gene Therapy of Arthritis and Related Disorders.
Clinical Treatments for End-Stage Osteoarthritis
The treatment of choice for end-stage osteoarthritis remains total joint replacement. To cater to the increased population of patients under the age of sixty years who are presenting with end-stage disease, considerable efforts have been made to design and track the clinical performance of implants intended to provide increased longevity and improved functionality. Implant modifications have focused on improving the wear resistance and mechanical properties of the bearing surfaces and on altering bearing surface geometry in order to improve range of motion.
High-Flexion Knee Designs
Total knee replacements intended to facilitate increased knee flexion have been available in the United States since 2002. In a biplane fluoroscopic analysis, presented at the recent meeting of the ORS, the degree of flexion afforded by a knee replacement designed for high-flexion activities was compared with that of a standard total knee replacement. No difference was found between the two designs in terms of the degree of flexion that was allowed clinically under passive motion and maximum forward lunge1. A more worrisome trend is the high prevalence of aseptic femoral component loosening that was reported in the study by Kang et al.2, in which 21% of the knees that had been implanted with an LPS-Flex Fixed Bearing knee (Zimmer, Warsaw, Indiana) were revised because of component loosening at five years postoperatively. In an analysis of seventy-two consecutive total knee replacements, the authors reported that the group that had loosening had a significantly greater amount of postoperative flexion as compared with the group that did not have loosening (136° compared with 125°). When a subset of patients were evaluated with imaging, the tibial component appeared to push the femoral component anteriorly off the femur as the knee was flexed past 130°. The authors concluded that although the high-flexion implant allowed for greater range of motion and more high-flexion activities than might be expected for a standard total knee replacement, the high rate of early femoral component loosening is of concern.
A symposium at the combined ORS/AAOS meeting highlighted potential advantages and disadvantages associated with modern metal-on-metal surface replacements. The importance of patient selection was emphasized, and it was suggested that as few as 6% of those who present for hip replacement are suitable candidates. With the correct indications, and careful surgical approach3, short-term data suggest that the longevity of surface replacements is similar to that of total replacements4. However, until long-term prospective randomized data become available, the longevity of the new generation of surface replacements in comparison with more traditional total joint replacement remains unknown.
Total Disc Replacements
Clinical follow-up studies on the performance of total disc replacements have focused on assessing the rate of revision when compared with fusion and on understanding the mechanism of failure of these relatively new implants. McAfee et al.5 reported a revision rate of 8.8% in a United States cohort of 589 patients who received the CHARITÉ lumbar Artificial Disc (DePuy Spine, Raynham, Massachusetts) as part of a multicenter prospective randomized United States Food and Drug Administration investigational device exemption study. The study included an additional group of ninety-nine patients who were managed with anterior spinal fusion with threaded cages; the revision rate for that group was 10%. The average time to revision was 423 days for the fusion group and 266 days for the group of patients with a total disc replacement. The clinical results from a multicenter study that followed 286 patients who had been managed with the ProDisc-L implant (Synthes Spine, Paoli, Pennsylvania) demonstrated significantly superior outcomes in terms of several parameters (neurological improvement, the SF-36 [Short Form-36] score, and the visual analog score at twenty-four months) as compared with circumferentially fused controls at a minimum of two years of follow-up6. A similar study of ninety-nine patients favored the Bryan cervical disc replacement over fusion controls when assessed according to improvements in the neck disability index, neck and arm pain, and the SF-36 score at the time of the two-year follow-up7. Data on the long-term performance of these implants are not yet available.
As presented at the most recent ORS meeting, retrieval analysis of revised disc replacements has identified polyethylene wear as a potential factor in long-term implant failure. Kurtz et al.8 reported that polyethylene oxidation was evident in forty SB CHARITÉ III total disc replacements (DePuy Spine) that were retrieved at an average of 7.2 years after implantation. Oxidation was more prevalent at the peripheral rim than in the central dome. Oxidation was also more severe in specimens that had been implanted for more than ten years than in those that had been implanted for less than ten years. Other studies by the same group of investigators demonstrated evidence of abrasive/adhesive wear of the polyethylene spacers, evidence of chronic rim impingement, and implant-site reactions, including inflammatory fibrosis and peri-implant osteolysis; in addition, a correlation was found between rim oxidation and polyethylene crack formation9,10. The authors suggested that long-term follow-up of patients managed with total disc replacement is required to monitor the status of the implants. Kelly et al.11 reported on nine ProDisc implants (four cervical and five lumbar) that had been retrieved after a maximum duration of implantation of 2.2 years. Despite the short duration of implantation, mild asymmetrical polyethylene insert wear and evidence of polished areas outside of the bearing surfaces consistent with metal-on-metal impingement were found. Bone growth onto <10% of the titanium-plasma-sprayed surfaces was also found. The authors concluded that obtaining additional similar data will help in the rational modifications in implant design to optimize the functionality of the devices.
The search continues for the ultimate bearing surface, with a goal toward reducing or eliminating osteolysis and related implant failure. The introduction of highly cross-linked ultra-high molecular weight polyethylene has undoubtedly demonstrated improved wear performance clinically and in vitro. However, the process of inducing high levels of molecular cross-linking by means of irradiation also can result in the generation of free radicals, which can lead to oxidation. To avoid oxidation, the irradiated polymer is typically heated (either above or below its melting point) to allow the free radicals to recombine and dissipate. This process can lead to a reduction in the mechanical properties of the polymer, for example, a reduction in fracture toughness, the clinical consequences of which are unknown. A particular area of recent research activity has been in efforts to modify highly cross-linked ultra-high molecular weight polyethylene so that the increased wear resistance provided by molecular cross-linking can be maintained without the reduced fracture resistance that accompanies cross-linking.
Three new manufacturing approaches are being explored. One involves the stabilization of free radicals through the impregnation of irradiated ultra-high molecular weight polyethylene with vitamin E12. Vitamin E protects polyethylene against oxidation, which renders the melting step that normally follows cross-linking with radiation unnecessary. Vitamin E also quenches free radicals, but it can decrease the cross-linking efficiency if it is present during irradiation. Using low concentrations of vitamin E and higher doses of radiation partially alleviates this problem. However, because some of the vitamin E is used up during irradiation, the long-term oxidative stability of irradiated blends is questionable. An alternative method being explored is to diffuse vitamin E into previously irradiated polyethylene while keeping the polyethylene at an elevated temperature. Again, the heating step may cause a decrease in mechanical properties, but it is necessary in order to ensure a homogeneous diffusion of vitamin E into the material.
A second approach involves sequentially irradiating and annealing polyethylene. Irradiation is conducted in three steps with interspersed annealing processes that together improve oxidative stability compared with that resulting from a single large dose of irradiation followed by annealing13. The third approach involves the photo-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) onto cross-linked polyethylene (CLPE)14. The concept is to create a hydrophilic layer with better wettability than a conventional polyethylene surface, thus increasing the chance for lubrication. Hip-simulator tests have shown that the graft polymerization material had minimal wear compared with untreated cross-linked polyethylene cups.
Regardless of the advances made in the design of total joint replacements, as the demographics of patients with end-stage osteoarthritis change, the need exists to establish a reliable method to treat young, active patients early in the course of the disease, thus delaying or eliminating the need for a total joint replacement. Toward this end, considerable advances have been made in understanding the genetic basis behind diseases of the musculoskeletal system and in developing ways to manipulate cells toward inducing a reparative response.
Molecular Medicine and the Genetic Basis of Musculoskeletal Diseases
Investigation of osteoarthritis as a genetic disease has led to increased research efforts in the area of epigenetics, as exemplified by a workshop on the topic during the 2008 annual meeting of the ORS (www.ors.org/web/Documents/Workshop_3_Handout.pdf). Epigenetic changes are defined as inheritable genetic alterations that do not involve DNA mutation. The process, which often involves methylation and/or acetylation of DNA that alters its ability to be transcribed, is well known to be an important mechanism during development. Its role in disease, particularly osteoarthritis, has only recently been investigated. As cited by workshop speaker David Hall, PhD, of the National Institutes of Health, Roach and Aigner15 recently reported that the increase in matrix metalloproteinase (MMP) expression in osteoarthritis is associated with altered methylation of key promoter sequences. Hall also presented data from his own laboratory showing that increasing levels of SIRT1, an enzyme involved in epigenetic gene silencing in osteoarthritis-affected chondrocytes, increased the expression of matrix genes and suppressed that of MMPs. Conversely, increasing levels of another such enzyme (HDAC2) had opposite effects, demonstrating the complex interactions that these enzymes likely coordinate. Future investigations will show whether epigenetic changes are indeed linked to the pathogenesis of osteoarthritis and whether enzymes such as SIRT1 and HDAC2 may be targets for therapy.
Obesity and Orthopaedics
Obesity continues to be a nationwide public health issue, and recent studies have revealed interesting interrelationships between energy metabolism and the biology of cartilage and bone. At Duke University, Farshid Guilak, PhD, directs a National Institutes of Health program project studying the role of obesity and inflammation in the pathogenesis of osteoarthritis. At the recent meeting of the ORS, his group reported on the rapid progression of osteoarthritis in both load-bearing and non-load-bearing joints (knees and temporomandibular joints, respectively) of mice that were fed high-fat diets16. These observations suggested that systemic factors may be more important than mechanical changes in the development of osteoarthritis in these animals. In contrast, this group reported on the absence of expected worsening of osteoarthritis in genetic models of mouse obesity (ob/ob and db/db mice), indicating that the systemic factors implicated in the high-fat diet studies may not relate to the leptin pathway.
The group led by Gerard Karsenty, MD, PhD, at Columbia University previously reported on the role of leptin in the central control of bone density. Last year, that group reported the novel finding that osteocalcin, a bone-specific matrix protein, is an essential regulator of systemic energy homeostasis. Previously, they had reported on the surprising absence of changes in bone mineralization in mice lacking osteocalcin. In a recent study, Lee et al.17 reported that mice deficient in osteocalcin displayed increased adiposity with attendant defects in the proliferation of pancreatic β-cells, insulin secretion, and insulin sensitivity. Osteocalcin also was shown to stimulate cyclin D1 and insulin expression in β-cells as well as adiponectin, an insulin-sensitizing adipokine, in adipocytes in vitro. Deletion of another osteoblast-specific gene that encodes a receptor-like protein tyrosine phosphatase termed OST-PTP had opposite effects on glucose metabolism, and this phenotype could be rescued by deleting one copy of osteocalcin. Together, these models indicate an important role of osteoblasts and osteoblast-specific proteins in energy metabolism and suggest novel targets in the treatment of obesity.
Bone Biology and Osteoporosis
Other noteworthy studies in the area of bone biology are elucidating new mechanisms of the control of bone density. In particular, the Wnt signaling pathway, which was highlighted in last year's Update, continues to be at the center of much research interest. An exemplary member of this signaling pathway is Sclerostin, a secreted molecule initially discovered as a bone morphogenetic protein (BMP) antagonist. Defects in the Sclerostin gene are responsible for sclerostosis, a condition characterized by dense bones and increased bone formation. As recently reported in the study by van Bezooijen et al.18 and reviewed in the February 2008 supplementary issue of this journal19, Sclerostin has been further characterized as a Wnt antagonist. On the basis of these activities, Amgen representatives reported at the recent meeting of the American Society for Bone and Mineral Research that they are developing an antibody to Sclerostin for the treatment of osteoporosis20. Their preclinical study in a primate ovariectomy model showed significant increases in bone density in association with the use of a monoclonal neutralizing antibody against Sclerostin.
Control of the Wnt pathway was a focus at the workshop on Wnt signaling at the 2008 annual meeting of the ORS (www.ors.org/web/Documents/Workshop_4_Handout.pdf). At that workshop, the group of Yingzi Yang, PhD, of the National Institutes of Health reported on their studies of transgenic mice in which they established that the activation of the canonical β-catenin pathway, which is responsible for many of the effects of Wnts, is downstream of signaling by hedgehog proteins, likely Indian hedgehog, during bone development. The hedgehogs are secreted growth factors, critical for embryonic patterning and development, that activate cells through binding and de-repression of cell-surface Patched receptors21. Interestingly, Yang's group showed that increased hedgehog signaling (through genetic deletion of one copy of the Patched gene) stimulated osteoprogenitor cells, yet paradoxically diminished bone density because of an increased osteoclastogenic capacity of the osteoprogenitors. Another transgenic mouse with diminished hedgehog signaling (through deletion of the co-receptor Smoothened) showed increased bone density suggestive of the counterintuitive possibility that suppression of osteoprogenitor cell activity may actually enhance bone density. The group of one of us (C.H.) also reported at the 2008 meeting of the ORS that an osteopenic phenotype could be induced in adult mice through gene-therapy-mediated overexpression of another hedgehog protein, Sonic hedgehog, by activation of the same mechanisms involving osteoprogenitor stimulation22. That study indicates that hedgehog-mediated mechanisms persist in postnatal animals, supporting the merit of further translational studies investigating the role of these pathways in the treatment of osteoporosis or other low-bone-density conditions.
Clinical studies in the area of bone density have shown an important side effect of antiresorptive therapy that should give surgeons pause in prescribing bisphosphonates to certain patients. Several groups, including that of Joseph Lane, MD, and Dean Lorich, MD, at the Hospital for Special Surgery in New York, have reported on the increased prevalence of subtrochanteric fractures in patients taking bisphosphonates for the long-term treatment of osteoporosis23-25. The Hospital for Special Surgery group described nontraumatic, nonpathologic fractures of the femoral shaft with a simple transverse pattern and hypertrophy of the diaphyseal cortex in patients taking Fosamax (Merck, Rahway, New Jersey). On the basis of those studies, the authors recommended that patients who have sustained a fracture should stop taking Fosamax and that patients receiving long-term treatment should be carefully monitored.
A major advance was made this year in the understanding of the genetic disease osteogenesis imperfecta. The group of Joan Marini, MD, PhD, at the National Institutes of Health has discovered that genetic defects that interfere with a pathway involving Prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CyPB) are the basis of a subtype of severe/lethal osteogenesis imperfecta. The P3H1, CRTAP, and CyPB form a complex in the endoplasmic reticulum that is essential for the normal hydroxylation of specific proline residues on type-I, and some other, collagens. In a recent article, Cabral et al.26 reported null mutations in the genes encoding CRTAP or P3H1 (called leprecan or LEPRE1 in humans) in several probands of patients with a subtype of severe/lethal osteogenesis imperfecta. Supporting these studies, a mouse deficient in CRTAP was previously shown to have a severe/lethal osteogenesis imperfecta phenotype27.
Degenerative Disc Disease
The intervertebral disc is a relatively isolated and poorly immunoreactive environment, which may be related to its tenuous blood supply. Human degenerative disc disease has early correlates of decreased disc hydration and blurring of the nucleus/anulus segments as well as later correlates of decreased disc height, proteoglycan loss, and increased gross vascularity. At the recent meeting of the ORS, Kwon et al.28 offered a potential mechanism for the latter stage of the degenerative process. Using in vitro studies of bovine intervertebral discs, they found that two factors capable of inducing angiogenic vessel growth (fibroblast growth factor and vascular endothelial growth factor) induced the expression of cartilage-degrading enzymes MMP-13 and ADAMTS-4. The investigators concluded that because these factors are also found within the degenerating disc, they may serve functions in both driving the vascular invasion of the disc and activating the enzymatic degradation of the disc cartilage matrix. Almost as an extension of these findings, Deng et al.29 reported that the disc matrix molecule biglycan may have an active role in controlling interleukin-1 (IL-1) signaling, and Hoyland et al.30 reported that IL-1β treatment of human disc tissue resulted in gene inductions for matrix metalloproteinases (ADAMTS-4, ADAMTS-9) and repression of tissue inhibitor of metalloproteinase-3. Those studies suggest a potential cycle of degeneration in which early destruction and loss of function of matrix molecules (biglycan) leads to lost repression of cytokine signaling, which in turn allows increased protease expression and amplified matrix destruction.
In an extension of previous work, Pichika and the group of investigators from Rush University31 reported that treatment of the rabbit intervertebral disc with osteogenic protein-1, four weeks after the initial anular puncture, resulted in a recovery of disc height loss, a reduction in cytokine expression (IL-1β, IL-6, tumor necrosis factor-α), and a reduction of matrix metalloproteinases (ADAMTS-4, ADAMTS-5). In a separate study, Masuda et al.32 reported similar in vitro anabolic effects of OP-1 treatment on proteoglycan synthesis, content, and turnover in cartilage explanted from human facet joints and concluded that OP-1 may delay or partially reverse the degenerative processes in the disc and facet tissues. Stem-cell-based interventions also were reported to have dramatic effects on degenerative disc disease. Bendtsen et al.33 reported on a porcine model in which the delivery of bone-marrow-derived stem cells to the disc space, twelve weeks after anular incision, resulted in an arrest of disc degeneration as assessed with magnetic resonance imaging. Similar findings were reported by Ganey et al.34 in a canine model involving the delivery of adipose-derived stromal cells, suggesting that the effect is generalizable. An interesting in vitro finding associated with the use of rat bone-marrow stem cells was that the acidity (pH) of the growth media greatly influenced gene expression and the proliferation and viability of the cells, which the authors suggested could limit their utility if implanted into discs of increased acidity as is seen in increasingly degenerate status35.
Regenerative Medicine for the Treatment of Musculoskeletal Diseases
A remarkable breakthrough was made in the area of stem cell and regenerative therapy this year, when two groups simultaneously reported on the feasibility of generating pluripotent stem cells—functionally similar to embryonic stem cells—from adult human fibroblasts. In separate experiments, the group of Shinya Yamanaka, MD, PhD36, at the Kyoto University in Japan and that of James Thomson, DVM, PhD37, at the Genome Center of Wisconsin showed that by using gene transfer to overexpress four transcription factors—Oct3/4, SOX2, Klf4, and c-Myc or Oct-4, SOX2, NANOG, and Lin28—adult human fibroblasts were programmed so that they behaved with the plasticity and proliferative potential of embryonic stem cells. The studies suggested that these “induced pluripotent stem cells,” or iPS, which can be propagated from a patient's own skin or other fibroblasts, could be used in place of embryonic stem cells for future regenerative medicine applications. Although promising, the use of iPS in humans requires further development to ensure safety. For example, c-Myc is associated with some tumors, so using methods that do not require it would be preferable.
In contrast to embryonic stem cells, bone-marrow-derived mesenchymal stem cells (BMSCs) have a safety profile that has allowed their use to progress to the clinical arena. Early studies suggested that BMSCs may be ideal for applications requiring bone or cartilage formation; however, clinical studies in osteogenesis imperfecta, for example, have not borne this out. Instead, recent studies have shown that BMSCs, delivered systemically, have a powerful anti-immunogenic activity that may be more important clinically than their ability to form connective tissues. In a recent report in The Lancet from the Developmental Committee of the European Group for Blood and Marrow Transplantation38, success in the use of bone-marrow-derived stem cells for the treatment of graft-versus-host disease was reported. These findings add to those of previous studies, dating back to 1995, on the successful use of these cells for the treatment of this disease.
The use of gene therapy continues to be developed for the treatment of arthritis, although not with genetic causes of the disease as a target. Instead, the gene-therapy strategies target inflammatory mediators and aim at producing high levels of antagonists to molecules such as TNF-α or IL-1. At the recent Gene Therapy of Arthritis and Related Disorders conference, studies focusing on the use of adeno-associated virus (AAV) were prominent. Because it does not induce a strong inflammatory response and because it transfers genes in a transient manner (thus avoiding the risk of insertional mutagenesis), AAV has become popular for orthopaedic applications and its intra-articular administration has been studied in clinical trials. At the meeting, representatives from Targeted Genetics (Seattle, Washington) reported resumption of their trial using AAV encoding the anti-TNF immunoglobulin for rheumatoid arthritis. Last year, the study had been halted because of the death of a study patient that has now been ascribed to disseminated histoplasmosis, a complication of rheumatoid arthritis that was not related to the gene therapy. Also at that meeting, Chris Evans, PhD, from Harvard University, reported that his group is now conducting toxicology studies on the use of AAV encoding the IL-1 receptor antagonist (IL-1RA) in preparation for a clinical study to treat osteoarthritis. IL-1RA is a naturally occurring molecule that antagonizes the inflammatory and catabolic actions of IL-1 in the joint, and retroviral delivery of this gene was the first orthopaedic gene therapy trial to have been conducted, also by Evans et al. Previously, the group of Edward Schwarz, PhD, at the University of Rochester reported on the use of AAV encoding a constitutively active form of activin receptor-like kinase-2 (ALK-2), a BMP receptor, to enhance the incorporation of massive grafts. In preparation for advancement to clinical trials with an innovative technique in which AAV is freeze-dried onto the allograft bone, the group has developed a noninvasive method involving the use of micro-computed tomography to determine graft incorporation and union to the surrounding bone39.
Enhancing the Treatment of Soft-Tissue Injuries
Challenges related to the treatment of sports-related or traumatic injuries of soft tissue include (1) regenerating native tissue that can function similarly to the original tissue and (2) restoring a functional osseous insertion site in a timely fashion. To face these challenges, advances have been made in understanding the cellular and molecular mechanisms that direct the healing response of soft tissue and its insertion site and the role that mechanical factors might play in directing the reparative process.
Ligament and Tendon
Recent studies have evaluated the role of the peripheral nervous system in ligament and tendon-healing. Neuropathic tissues, such as in patients with diabetes mellitus, are known to have poor healing potential, suggesting an important role for neurogenic factors in connective tissue biology. The peripheral nervous system affects tissue function by the action of neuropeptides, which are a class of chemicals that influence cell proliferation, local blood flow, vascular permeability, and inflammatory cell chemotaxis. The effect of neuropathy on healing of the medial collateral ligament was studied in rats that had undergone surgical sympathectomy (autonomic neuropathy) or femoral nerve transaction (sensory neuropathy)40. There were significant decreases in various neuropeptides, such as substance P, calcitonin gene-related peptide, and vasoactive intestinal peptide, in the denervated tissues. Impaired healing, demonstrated by significant decreases in failure force of the healing ligaments, was seen in both groups of rats. The investigators went on to demonstrate that local delivery of specific neuropeptides by means of mini-osmotic pumps could reverse the functional deficits of these neuropathic ligaments. Local delivery of substance P and vasoactive intestinal peptide improved ligament-healing, with failure forces higher than the force required to rupture a normal, uninjured medial collateral ligament. The improvements in ligament strength were accompanied by improved cell alignment and extracellular matrix organization. That study was the first, to our knowledge, to demonstrate the recovery of strength to the level of an intact ligament.
Another study examined the relationship between ligament-healing and nerve ingrowth with use of a rat Achilles tendon-healing model41. There was improved healing in rats that were subjected to increased physical activity (wheel running) as compared with rats with normal cage activity and rats that were immobilized in a plaster cast. Conversely, the levels of calcitonin gene-related peptide were lower in the most active group. Although it is possible that physical activity could inhibit new nerve ingrowth, the positive effects of neuropeptides on cell proliferation combined with the accelerated healing in the animals with increased physical activity suggest that a higher level of activity results in earlier nerve ingrowth and then disappearance from the healing tendon. It is known that innervation of the Achilles tendon is normally confined to the paratenon and that new nerve fibers grow into the tendon proper after rupture but disappear after healing. These findings suggest that activity level affects the rate of neuronal plasticity and thus tendon-healing.
Anterior Cruciate Ligament
The anterior cruciate ligament continues to be an area of intense investigation. It is well established that the intra-articular anterior cruciate ligament has very little healing potential, in contrast with extra-articular ligaments such as the medial collateral ligament. Healing in extra-articular ligaments begins with the formation of a fibrin clot that acts as a scaffold to bridge the ends of the torn ligament. It is believed that a key factor in the inability of the anterior cruciate ligament to heal relates to the premature loss of the provisional fibrin clot scaffold due to plasmin in the synovial fluid. This hypothesis is supported by ongoing work in the laboratory of Murray and colleagues at Harvard Medical School. Those investigators found that healing of a central defect in the canine anterior cruciate ligament could be improved with use of a collagen-platelet-rich plasma hydrogel in the wound site42. Treatment of the intra-articular wound with a collagen-platelet-rich plasma hydrogel resulted in increased filling of the wound site with repair tissue. Furthermore, this repair tissue had growth-factor and protein-expression profiles similar to those seen in a healing extra-articular ligament (the medial collateral ligament). In a further extension of this work, the authors performed bilateral anterior cruciate ligament transections in Yorkshire pigs43. In each animal, one side was treated with suture repair alone whereas the other side was treated with suture repair and augmented with the placement of a collagen-platelet-rich plasma hydrogel at the anterior cruciate ligament transection site. The authors found significant improvements in load at yield, maximum load, and linear stiffness at four weeks in the ligament treated with the collagen-platelet-rich plasma hydrogel. That study suggests that there is adequate intrinsic cellular and vascular response in the injured anterior cruciate ligament to support functional healing if an appropriate scaffold is provided. Further extension of this work may ultimately allow repair of the anterior cruciate ligament instead of replacement with a tendon graft.
Another avenue that has been explored within the ligament field has been related to tissue-engineered substitutes for the anterior cruciate ligament. Recent work has defined the fundamental design requirements that must be met44. A ligament replacement must allow for osseous attachment with use of standard techniques, and the graft must match the material properties (stiffness and yield load) of the native anterior cruciate ligament. Any new material must avoid the problems associated with previous ligament-replacement devices, such as abrasion, production of particulate debris, stress-shielding of newly-formed tissue, and fatigue failure. The material should be bioabsorbable and should allow cellular infiltration, cell differentiation, and new matrix formation. Most importantly, the design must anticipate the changes that occur with resorption of the material and tissue ingrowth, so that load is gradually transferred to the developing neoligament and stress-shielding of the newly formed tissue is avoided.
Although a range of biomaterials and graft constructs have been explored in the past, silk recently has been identified as a promising material for a ligament scaffold. A collaborative effort between a commercial group and orthopaedic surgeons44 is exploring silk fibroin derived from Bombyx mori. This material undergoes bioresorption through the same mechanism as collagen. The graft substitute has been designed with a number and geometry of bundles so as to provide an initial tensile strength of 2200 N while also allowing transport of nutrients into the material after implantation. This material has been tested in a goat anterior cruciate ligament reconstruction model. At twelve months after implantation, there was no evidence of synovitis or particulate debris in the joint, and the cartilage surfaces were normal. There was satisfactory knee stability (as determined with the Lachman examination). Histologic analysis demonstrated that the material was undergoing resorption and that there was deposition of organized collagenous tissue with aligned fibroblasts in the graft. The well-aligned matrix that was forming suggests that the designed-in graft stiffness allowed appropriate stress transfer to the developing tissue, avoiding stress-shielding. Stiffness and ultimate tensile strength increased over time, returning to native levels by twelve months. These results support the concept of using this silk fibroin material for ligament tissue engineering.
Another advance in the field of ligament tissue engineering was made by Dr. Helen Lu and colleagues at Columbia University. Those investigators are studying methods to engineer the complex structure and composition of the ligament-to-bone insertion site. The native insertion site contains a fibrocartilage zone between ligament and bone, which does not reproducibly form following standard tendon graft reconstructions. These investigators are using a co-culture model that permits osteoblast-fibroblast interactions, through both physical contact and paracrine interaction, that mimics the in vivo condition in which a tendon graft is placed in a bone tunnel45. The co-culture induced fibroblast-mediated mineralization and resulted in the expression of interface-relevant markers such as type-II collagen and aggrecan. These findings suggest that osteoblast-fibroblast interactions may lead to cell differentiation and eventual fibrocartilage formation and may provide new insight into the mechanism of fibrocartilage formation, which is critical for interface tissue engineering.
A related area of investigation centers on rotator cuff tendon-to-bone healing, which is known to have relatively high failure rates following surgical repair. Investigators at Washington University have used a mouse model to study the development of the rotator cuff tendon-to-bone insertion as this information may ultimately lead to methods to improve rotator cuff tendon-healing. In one study, investigators reported that decreased muscle-loading delays maturation of the tendon insertion site during postnatal development46. An intramuscular injection of botulinum toxin A was used to paralyze the supraspinatus muscle at birth. By twenty-one days, fibrocartilage development was delayed, mineralized bone was decreased, and osteoclast activity was higher in the botulinum-treated group as compared with the contralateral, saline-solution-treated control group. These results indicate that the development of the tendon insertion is sensitive to its mechanical environment. A reduction in muscle-loading delayed the development of the tendon-to-bone insertion site by impeding the accumulation of mineralized bone and fibrocartilage formation. In a related study, those investigators localized the expression of several extracellular matrix and growth-factor genes during insertion site development47. Mouse embryos and neonatal mice were killed and examined with use of in situ hybridization. Fibroblasts of the supraspinatus tendon expressed type-I collagen at all time-points. Type-II collagen was first expressed by chondrocytes in the fibrocartilage and mineralized fibrocartilage at seven days, but the interface between tendon and bone did not form into a mature fibrocartilaginous insertion until twenty-one days postnatally, suggesting that the mechanical environment plays an important role in the formation of this tissue. Type-X collagen was first expressed by the chondrocytes in the mineralized fibrocartilage at fourteen days and persisted in the mineralized fibrocartilage at fifty-six days. Type-X collagen expression may play a critical role in the development of mineralized fibrocartilage, and its expression is notably absent or diminished during adult tendon-to-bone healing. Transforming growth factor-β3 (TGF-β3), a growth factor that is expressed during “scarless” healing in fetal wounds, was expressed up until 15.5 days after conception. After this time-point, TGF-β1 was expressed. This time-point coincides with the stage in fetal development at which wounds heal through scarring rather than regeneration of normal tissue. TGF-β1, but not TGF-β3, is expressed in adult tendon-to-bone healing. These findings suggest the possibility that TGF-β3 could improve tendon-to-bone healing.
In summary, as implant designs are modified to adapt to the changing demographics of those presenting with end-stage musculoskeletal degeneration, and the molecular and genetic basis for skeletal diseases begins to be elucidated, the possibility of a continuum of treatment from the early to the end stages of diseases seems more plausible. The challenges that lie ahead will be focused on translating the molecular and genetic findings into clinically viable, safe, and efficacious treatments.
NOTE: The authors acknowledge the contributions of their colleagues from the Research Division at the Hospital for Special Surgery to this manuscript. In particular, the authors thank Dr. Adele Boskey, Dr. Timothy Wright, and Joe Lipman for their input.
Specialty Update has been developed in collaboration with the Council of Musculoskeletal Specialty Societies (COMSS) of the American Academy of Orthopaedic Surgeons.
Disclosure: The authors did not receive any outside funding or grants in support of their research for or preparation of this work. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the authors, or a member of their immediate families, are affiliated or associated.
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