Basic science studies remain a critically important part of the knowledge-discovery process in musculoskeletal patient care. This article highlights several of the key advances in orthopaedic basic science over the past year. It should be noted that some topics of tremendous basic science interest, most notably orthopaedic infection and neoplasia, are not covered in detail in this article because they have been the focus of previous Specialty Update articles in The Journal1,2.
Joints and Joint Disease
A Joint as an Organ
It is now widely accepted that a joint is best considered an organ, rather than a simple tissue, and that osteoarthritis (OA) represents a spectrum of pathologies affecting several of the core structures that make up the joint. Early understanding of OA focused on the changes that were grossly evident on joint inspection, including cartilage degradation, subchondral bone sclerosis, synovial inflammation, and osteophyte formation. More recently, this picture has been expanded to include the contributions of local changes in peri-articular muscles, nerves, and intra-articular structures such as the fat pad. This expanded understanding of the complexity of joint structure and function3 should facilitate the development of more robust and productive in vitro models for studying joint disease and its management. It will also drive the development of more effective regenerative strategies that target multiple aspects of organ function, rather than focusing on cartilage repair/regeneration alone.
The majority of preclinical, induced (i.e., nonnatural) models of OA involve the creation of traumatic instability within the joint. Although this may limit the translational relevance of studies performed with these models, particularly with regard to the efficacy of pharmacological or biological therapies, it has facilitated notable recent advances in our understanding of the effects of trauma on function of the joint organ. Blaker et al. reported on the use of mouse models of posttraumatic OA (PTOA) and recommended a classification scheme that incorporates 3 types of lesions based on the extent of the loading that initiates the damage (type I: loading that initiates immediate structural damage and instability, type II: moderate loading with no immediate instability, and type III: minor loading with no immediate instability)4. Mouse models exist for all of the injury types, opening up the potential for detailed and comparative studies between different degrees of injury and, in particular, for teasing apart the effects of acute versus chronic instability.
Martin et al. recommended the use of subclassification in their work on the development of predictive models for PTOA5. They identified 3 broad categories of models that can be used to examine the interactions between mechanical overloading and cartilage loss. At one end of the spectrum are the explant and organ (joint) culture models, which allow for very precise measurement of the cellular response to controlled joint loading. In the middle are the animal models, in which it is possible to study the effects of trauma and instability on joint structure and function and to evaluate the safety and efficacy of candidate interventions. At the other end of the spectrum lies in silico modeling, by which patient-specific computer models, built using data from the other 2 types of models, can be employed to predict the likely response to injury and the most appropriate treatment approach.
Cartilage Repair and Regeneration
There is a clear distinction between acute osteochondral injuries, most often the result of trauma, and chronic damage that is often associated with OA. This difference is likely to be of great importance in the context of developing biological strategies for repairing or replacing cartilage that has been lost or damaged, as the microenvironment into which biologics are injected/implanted may be very different in acute versus chronic settings. Improved understanding of normal cartilage development and homeostasis is critical to the development of optimal regenerative strategies. For example, the recognition that articular cartilage and synovium share common precursors has fueled interest in synovium itself as a source of cells for transplant into osteochondral defects6.
Jiang and Tuan recently reported on the identification of cartilage stem/progenitor cells (CSPCs) that appear to be capable of self-repair7. These stem-like cells have been found in adult human, bovine, and equine cartilage, suggesting that they represent a biologically conserved strategy for joint homeostasis. The proliferation of CSPCs in cell culture can be selectively enhanced through careful control of the culture conditions, with 2-dimensional, low-density culturing appearing to be most favorable. CSPCs were found to be capable of forming hyaline cartilage explants in vivo using a mouse model, and a follow-up study involving 15 human patients with isolated osteochondral lesions produced encouraging results8.
Advances in cell therapies for cartilage repair continue to move in parallel with developments in biomaterials for delivering and/or retaining the cells at the site of implantation. Current therapies, whether involving local stimulation with microfracture or direct replacement with cartilage transplant, appear well suited for small, isolated defects. The management of larger, less constrained defects remains more challenging and is perhaps the area where regenerative medicine has most to offer. Much of the emphasis remains on bioengineering of scaffold structures capable of resurfacing large areas of the joint, an area of investigation that has seen major advances. In some cases, for example, involving the meniscus, it may be sufficient to rely on the biomaterial alone for replacement of the structure9, but for articular cartilage, a seeded construct may represent a better approach. Moutos et al. used a woven, anatomically contoured scaffold to support the directed expansion of adipose-derived stem cells, leading to the formation of a robust hyaline cartilage that may find clinical utility in resurfacing the femoral head10.
Crosstalk Among Tissues
Interactions Between Bone and Muscle
The effects of direct muscle forces on bone mass and bone structure have been studied for many years, but it is increasingly apparent that there are substantial muscle-bone interactions that are not based on mechanical effects11. The recognition of both bone and muscle as secretory, endocrine organs has changed the way that we think about muscle-bone interactions. Starting with myostatin, the list of muscle-derived chemokines, or myokines, continues to expand, and it has been suggested that these molecules may play a pivotal role in the health-related benefits of physical exercise on cardiac, brain, renal, and hepatic function12. Recent work, much of it based on studies of genetically modified mice, has established that both fibroblast growth factor 23 (FGF23) and osteocalcin, produced within bone, exert important remote effects. Deletion of FGF23 expression within bone leads to disorders of phosphate metabolism13, and deletion of osteocalcin produces disturbances in energy metabolism through effects on glucose metabolism14.
Interactions Beyond Muscle
Across medicine, there is a growing interest in the role of the central nervous system and its associated neuropeptides on the function of other organs. Leptin, first identified as a modulator of appetite and satiety, has also been shown to exert influence on bone mass both remotely, via direct effects on bone cells, as well as centrally in the brain15. The sympathetic and parasympathetic components of the autonomic nervous system have also come under increasing scrutiny, both for their sensory functions and for their effector functions in linking the brain and the skeleton16. Much remains to be determined regarding the specifics of these interactions, but it is clear that further investigation of these pathways will be illuminating in terms of both improving our understanding of bone disease and identifying potential targets for therapy.
Interactions between bone cells and cells of the immune system have been a topic of great interest for several years, with the initial focus being on the role of T cells in inflammatory bone and joint conditions. Recognition of the hematopoietic stem-cell niche, and the role played by bone cells in establishing and maintaining it, served to further highlight the importance of bone-immune cell interactions in vivo. Recent work has identified the importance of T cell-mediated interactions in defining skeletal responsiveness to parathyroid hormone17 and the potential role of activated natural killer (NK) cells in controlling osteoclastic activity within inflammatory bone lesions18.
Bone and the Microbiome
In the context of remote regulators of bone turnover, another more recent area of research interest is the influence of gut microbiota19. The microbiome has been shown to be a key regulator of many homeostatic processes that occur outside the gastrointestinal tract, including important effects on host immunity, and studies in mice have shown that the gut microbiota can influence bone mass. This has led to interest in the use of probiotics to modulate bone mass20,21, although much remains to be determined in order to identify the optimal combination of probiotic, timing, and duration of treatment.
Mechanism(s) of Action of Adult Stem Cells
Stem cells remain one of the most exciting potential vehicles for delivering therapy in musculoskeletal disease. Techniques for isolating, expanding, and characterizing adult stem cells from bone marrow, adipose tissue, cord blood, peripheral blood, or synovium continue to evolve, but the relative scarcity of viable progenitor cells in the source tissue remains a major challenge. Several recent reports have presented potential approaches to improving the clinical utility of mesenchymal stem cells (MSCs) as effectors of musculoskeletal tissue regeneration.
Sheyn et al. made use of recent advances in somatic cell reprogramming to generate inducible pluripotent stem cells (iPSCs) and then differentiated them into MSCs using transforming growth factor-beta22. The induced MSCs were found to be pluripotent in vitro and were more effective than bone marrow-derived, noninduced MSCs transduced with the gene for bone morphogenetic protein-6 in repairing radial defects in mice.
The use of synovial-derived MSCs was studied in 2 recent reports. In the first, human cells were injected into the knee joints of rats that had undergone transection of the anterior cruciate ligament 1 week previously23. A single injection of cells was found to be ineffective in modulating the progression of degenerative joint disease, but repeated weekly injections were found to be chondroprotective over a 12-week period. Using immunohistochemistry, those authors demonstrated that the majority of the injected cells remained undifferentiated in vivo, functioning instead as local sources for chondroprotective proteins and anti-inflammatory cytokines. The second study explored the use of autologous synovial-derived MSCs in the regeneration of meniscal defects in a nonhuman primate model24. MSC aggregates were implanted directly into defects in the anterior half of the medial meniscus, and the animals were followed for either 8 or 16 weeks. The reparative response in the meniscus, and any attendant changes in the adjacent articular cartilage, were assessed by magnetic resonance imaging (MRI), gross observation, and histopathology. In this model, MSCs promoted meniscal regeneration and delayed the progression of cartilage damage and OA.
The Role of Clock Genes and Diurnal Variation in Gene Expression
The importance of diurnal variation has been established in many areas of musculoskeletal biology, including bone turnover. Previous work showed that the loss of clock genes eliminates the ability of bone to respond to leptin25, but more recently, attention has moved to looking at the clock genes in the context of articular cartilage. Dudek at al. reported that targeted disruption of the clock transcription factor BMAL1 abolished normal circadian expression of a number of genes associated with cartilage homeostasis26. BMAL1 expression was also reduced in cartilage explants from humans with OA and from aged mice, implicating BMAL1 as an important potential factor in OA progression. Guo et al. found that the regulation of clock-controlled genes is itself disrupted by catabolic cytokines implicated in cartilage degradation27. In the context of spinal disease, an interesting study of rats demonstrated that passive exposure to cigarette smoke resulted in a pronounced (6 to 9-hour) change in the activity of clock genes in the intervertebral disc28.
Gene Editing with CRISPR-Cas9
Selective gene targeting using CRISPR (clustered regularly interspersed short palindromic repeat) technology and the RNA-guided nuclease, Cas9, has rapidly become the predominant gene-editing tool in genomics. In the past year, there were a number of high-profile and very exciting reports on the use of CRISPR-Cas9 gene editing in the study of musculoskeletal disorders. In Duchenne muscular dystrophy, a series of 3 complementary studies demonstrated the successful use of this technology to restore dystrophin expression in a mouse model of the disease29-31. There has also been recent work on the use of this technology to target musculoskeletal cancers such as osteosarcoma32.
Metabolomics and the Identification of Disease Signatures and Therapeutic Targets
The use of wide-scale screening of tissue samples using metabolomics has produced interesting observations across a range of orthopaedic conditions. Mickiewicz et al. reported on the use of metabolomics to identify a potential signature for osteoarthritis33. Liu et al. compared metabolomic profiles in plasma samples from 30 clinical patients having osteonecrosis of the femoral head with those from 30 control patients, finding that 123 metabolites were differentially expressed in the 2 populations34. Interestingly, many of these candidate markers were involved in lipid, glutathione, and energy-associated pathways that might be associated with inflammation, oxidative stress, and energy deficiencies that have been implicated in the pathogenesis of osteonecrosis. In a mouse model, Shum et al. used metabolomics to demonstrate that mitochondrial oxidative function is impaired in aged animals and that the negative effects of aging on bone formation, bone mass, and bone strength can be partially reversed by targeting cyclophilin D, a key determinant of mitochondrial function35.
Animal Models and Their Reproducibility
The past decade has seen a notable increase in public awareness regarding the contentious issue of animal use in biomedical research. One of the fundamental tenets of animal research is that the “benefit” (to patients and society at large) should exceed the “cost” to the animal in terms of pain and distress. As a result of improvements in the level of detail at which researchers and clinicians are now able to characterize disease processes, it is becoming evident that many of the induced disease models that are used routinely fail to completely replicate the pathology that is seen in the clinic. More disturbing is the suggestion that, even when validated animal models are used, the variability in how animal models are used means that it is often impossible to make direct comparisons between studies performed at different institutions or by different investigators. The National Institutes of Health has identified irreproducibility as a major obstacle to scientific advances36 and, according to a recent report, this problem of irreproducibility costs approximately $28 billion annually in the United States37.
Arguments are being made that the “3Rs” (replacement, reduction, and refinement) should now be expanded to the “4Rs” or even the “5Rs” to incorporate the need for reproducibility and (clinical) relevance of an animal model. In the meantime, the orthopaedic research community is encouraged to work collectively to identify and propagate best practices in the use of animal models in its work38. Journals will continue to play a pivotal role in ensuring the integrity of this process by requiring that authors (1) verify that the work has been approved by the relevant national and/or institutional regulatory authorities, and (2) provide enough specific detail in the “Methods” section to ensure that the work can be replicated by other laboratories.
Upcoming Meetings and Events Related to Orthopaedic Basic Science
* The Orthopaedic Research Society 2017 Annual Meeting will be held March 19-22, 2017, in San Diego, California (http://http://www.ors.org/2017annualmeeting/).
* The Gordon Research Conferences “Cartilage Biology & Pathology: Understanding Biology to Achieve Better Cartilage Health” will be held April 2-7, 2017, in Barga, Italy (https://http://www.grc.org/programs.aspx?id=13112).
* The Bone Research Society 2017 Annual Meeting will be held June 25-27, 2017, in Bristol, United Kingdom (http://boneresearchsociety.org/meeting/bristol2017/).
* The European Society of Biomechanics 2017 Annual Meeting will be held July 2-5, 2017, in Seville, Spain (https://esbiomech.org/).
* The American Society for Bone and Mineral Research 2017 Annual Meeting will be held September 9-12, 2017, in Denver, Colorado (http://http://www.asbmr.org/Default.aspx).
* The European Orthopaedic Research Society 25th Annual Meeting will be held September 13-15, 2017, in Munich, Germany (http://http://www.eors2017.org/).
NOTE: The author thanks Farsh Guilak, PhD (Orthopaedic Research Society President, 2016-17), and Martin Stoddart, PhD (Chair, ORS Basic Science Education Committee), for critical input regarding the selection of topics for this review.
Investigation performed at the Surgical Discovery Centre, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
Specialty Update has been developed in collaboration with the Board of Specialty Societies (BOS) of the American Academy of Orthopaedic Surgeons.
Disclosure: The author reports a stipend from JBJS for manuscript preparation of this article. The Disclosure of Potential Conflicts of Interest form is provided with the online version of the article.
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