Musculoskeletal Research: Current Methods and Innovative Approaches

Musculoskeletal disorders afflict millions of people and are a leading cause of disability worldwide¹. Despite that, musculoskeletal disorders are understudied and less well understood compared to other prevalent chronic disorders (e.g., cancer and diabetes). Furthermore, the prevalence of disabilities associated with musculoskeletal disorders is projected to increase, which underscores the importance of research investigating the molecular mechanisms and cellular pathways that underpin the development and progression of musculoskeletal diseases. Accordingly, there is an increasing need for methodologies that allow for a better understanding of musculoskeletal diseases on the molecular level and facilitate the development of innovative therapeutics.

Peripheral nerve injury is associated with ~3% of trauma cases and is a common cause of morbidity in orthopedic trauma patients². However, a precise treatment that rescues the nerve functions and reverses the muscle atrophy associated with traumatic peripheral nerve injury (TPNI) remains elusive³. Therefore, studies in murine models are instrumental for evaluating the efficacy of investigational TPNI treatments; however, the field lacks predictable nerve transection animal models that allow reliable functional recovery measurements or characterization of nerve regeneration. In their study included in this methods collection, Lee et al. describe a TPNI model that ensures a fixed injury pattern and facilitates the measurement of functional recovery⁴. In general, functional recovery following TPNI depends on the severity of the injury, and a major advantage of the protocol described by Lee et al.⁴ is the use of a calibrated digital device that monitors the pressure variation in real time during the nerve crushing surgery. This limits the variability in the severity of the induced injury and, subsequently, the recovery.

Osteoarthritis (OA) is the leading cause of disability affecting the lower extremities in older adults and, thus, constitutes a major societal and economical burden⁵. Murine models that mimic the phases of OA development exist, including the widely used destabilization of the medial meniscus (DMM) model of post-traumatic OA^6,7. The article by Dunning et al. introduces a destabilization and cartilage scratch (DCS) model of OA, which is a modified version of the DMM model and involves imparting further damage to the cartilage by introducing three scratches to the cartilage surface⁸. The DCS model represents an accelerated model of OA and is suitable for studies focusing on osteophyte formation, synovial inflammation, bone remodeling, and pain measurements during the early phases of OA. The model also recapitulates the damage inflicted directly on the cartilage during arthroscopic surgeries and other clinical interventions.

Several mechanistic studies that are infeasible in mice can be performed in cultured cells, which underscores the importance of in vitro experiments that complement in vivo studies. Primary cells are an important research tool as they recapitulate the endogenous, in vivo conditions more faithfully than stable cell lines. Accordingly, developing protocols that facilitate the isolation of viable primary cells from different musculoskeletal tissues is essential to advance the research in this field. The paper by Jagadeeshaprasad et al. details a methodology for the enzymatic and mechanical digestion of human newborn foreskin to simultaneously isolate three types of primary cells: keratinocytes, fibroblasts, and Schwann cells (SCs)⁹. The isolation of SCs from skin represents a major advancement that this protocol introduces to the well-established protocols for isolating keratinocytes and fibroblasts. The protocol facilitates the co-culture of the three cell types, which is essential for studies investigating the potential roles of SCs in modulating the homeostasis and functions of keratinocytes and fibroblasts.

Overexpression and knockdown are commonly used approaches to study gene functions. Although these experiments are easily and routinely conducted in cell cultures in vitro, it is more challenging to manipulate gene expression in vivo due to the challenges in achieving the efficient delivery of plasmid DNA or small interfering RNA to the target cells/tissue. Hain and Waning describe an easy protocol to electroporate DNA plasmid into mouse skeletal muscle to overexpress the encoded protein in the myofibers¹⁰. A major drawback of the existing protocols for injecting or electroporating DNA plasmids into skeletal muscles is the negative impact of these procedures on muscle contractility. The protocol introduced by Hain and Waning¹⁰ addresses this limitation as it does not compromise the skeletal muscle contractility, which allows the assessment of muscle contractility as a functional outcome of varying protein expression.

Micro-computed tomography (µCT) is an imaging modality that is commonly used to assess the three-dimensional (3D) structure of bone¹¹. µCT is the gold standard for human bone imaging in the clinic and is very widely used in evaluating bone structure and bone regeneration in preclinical studies¹². µCT involves a large number of X-ray images obtained from mineralized bone, which inherently has excellent contrast to X-ray. When used to assess bone formation during the fracture healing process, µCT provides detailed information about the 3D structure and density of the healing callus. Although the quantitation of intact bone using µCT is well established, there is a lack of consensus on a µCT protocol for the assessment of the fracture callus. Wee et al. provide a standardized, step-by-step protocol for accurate assessment of the fracture callus during different phases of bone fracture healing¹³. The protocol utilizes a state-of-the-art analysis platform that enables image visualization, re-alignment, and callus segmentation. This contribution will help establish a more standardized and consistent approach for fracture callus analysis across the field.

In summary, this methods collection presents experimental protocols used in a wide array of musculoskeletal tissues: bone, cartilage, peripheral nerve, skeletal muscle, and skin. We hope that this collection proves useful for newcomers as well as experts in the fast-growing field of musculoskeletal research. The future of the field will be greatly enhanced by the continuous efforts aiming at developing novel research methodologies. Endeavors to establish novel murine models for musculoskeletal disorders and to develop innovative approaches for the local and targeted delivery of therapeutics to musculoskeletal tissues are especially valuable.

The authors have nothing to disclose.

This work was supported by the National Institutes of Health (NIH), grant R01 DK121327.