A two-stage digestion of whole murine knee joints for single-cell RNA sequencing.

Objective: Single-cell RNA sequencing (scRNA-seq) is a powerful technology that can be applied to the cells populating the whole knee in the study of joint pathology. The knee contains cells embedded in hard structural tissues, cells in softer tissues and membranes, and immune cells. This creates a technical challenge in preparing a viable and representative cell suspension suitable for use in scRNA-seq in minimal time, where under-digestion may exclude cells in hard tissues, over-digestion may damage soft tissue cells, and prolonged digestion may induce phenotypic drift. We developed a rapid two-stage digestion protocol to overcome these difficulties. Design: A two-stage digest consisting of first collagenase IV, an intermediate cell recovery, then collagenase II on the remaining hard tissue. Cells were sequenced on the 10x Genomics platform. Results: We observed consistent cell numbers and viable single cell suspensions suitable for scRNA-seq analysis. Comparison of contralateral knees and separate mice showed reproducible cell yields and gene expression patterns by similar cell-types. A diverse collection of structural and immune cells were captured with a majority from immune origins. Two digestions were necessary to capture all cell-types. Conclusions: The knee contains a diverse mixture of stromal and immune cells that may be crucial for the study of osteoarthritis. The two-stage digestion presented here reproducibly generated highly viable and representative single-cell suspension for sequencing from the whole knee. This protocol facilitates transcriptomic studies of the joint as a complete organ.

Regional and disease-related differences in properties of the equine temporomandibular joint disc.

Temporomandibular joint (TMJ) disorders affect up to 12% of the human population, and naturally occurring TMJ diseases are increasingly recognized in animals. The TMJ disc plays a major role in TMJ disorders in people, but little is known about its role in TMJ pathology in animals. This study characterizes differences in properties of equine TMJ discs associated with age, disc region, and presence of TMJ osteoarthritis (OA). Discs were dissected from both TMJ's of sixteen horses euthanized for reasons unrelated to this study. Each joint was grossly evaluated and scored as normal, mild OA, or severe OA. Samples from the rostral, caudal, lateral, central, and medial regions of the disc were subject to compressive testing, quantitative biochemistry, and histology. Samples from the lateral, central, and medial region were tested for tensile properties in the rostrocaudal and mediolateral directions. We found that the equine TMJ disc is highly anisotropic, and its glycosaminoglycan (GAG) content and compressive stiffness vary between disc regions. The disc also exhibits increasing GAG content and compressive stiffness with increasing age. While equine TMJ disc properties are generally similar to other herbivores, greater compressive stiffness throughout the disc and greater GAG content in its rostral region suggest that mechanical demands on the TMJ disc differ between horses and other species. Importantly, a region-specific decrease in compressive stiffness was observed associated with joint disease and corresponded to cartilage erosions in the underlying condylar surface.

Biomechanical evaluation of two plating configurations for critical-sized defects of the mandible in dogs.

OBJECTIVE To compare the biomechanical behavior of mandibular critical-sized defects stabilized with 2 plating configurations under in vitro conditions resembling clinical situations. SAMPLE 24 mandibles harvested from 12 adult canine cadavers. PROCEDURES 8 mandibles were kept intact as control samples. A critical-sized defect was created in 16 mandibles; these mandibles were stabilized by use of a single locking plate (LP [n = 8]) or an LP combined with an alveolar miniplate (LMP [8]). Mandibles were loaded in cantilever bending in a single-load-to-failure test with simultaneous recording of load and actuator displacement. Stiffness, yield, and failure properties were compared among groups. Mode of failure was recorded. Radiographic evidence of tooth root and mandibular canal damage was quantified and compared between groups. RESULTS Stiffness and yield loads of single LP and LMP constructs were < 30% of values for intact mandibles, and failure loads were < 45% of values for intact mandibles. There were no consistent biomechanical differences at failure between single LP and LMP constructs, but the LMP construct had greater stiffness and strength prior to yield. Frequency of screw penetration of teeth and the mandibular canal was significantly greater for LMP than for single LP constructs. CONCLUSIONS AND CLINICAL RELEVANCE Both fixation methods were mechanically inferior to an intact mandible. The LMP construct was mechanically stronger than the LP construct but may not be clinically justifiable. Addition of an alveolar miniplate provided additional strength to the construct but resulted in more frequent penetration of tooth roots and the mandibular canal.

Characterization of the Temporomandibular Joint of Southern Sea Otters (Enhydra lutris nereis).

The structure-function relationship of the temporomandibular joint (TMJ) of southern sea otter has largely not been described. This study aims to describe the histological, biochemical, and biomechanical features of the TMJ disk in the southern sea otter. The TMJ disks from fresh cadaver heads of southern sea otter adult males (n = 8) and females (n = 8) acquired from strandings were examined. Following macroscopical evaluation, the TMJs were investigated for their histological, mechanical, and biochemical properties. We found that the sea otter TMJ disks are, in general, similar to other carnivores. Macroscopically, the TMJ disk was highly congruent, and the mandibular head was encased tightly by the mandibular fossa with a thin disk separating the joint into two compartments. Histologically, the articular surfaces were lined with dense fibrous connective tissue that gradually transitioned into one to two cell thick layer of hyaline-like cartilage. The disk fibers were aligned primarily in the rostrocaudal direction and had occasional lacuna with chondrocyte-like cells. The disk was composed primarily of collagen type 1. Biochemical analysis indicates sulfated glycosaminoglycan content lower than other mammals, but significantly higher in male sea otters than female sea otters. Finally, mechanical analysis demonstrated a disk that was not only stronger and stiffer in the rostrocaudal direction than the mediolateral direction but also significantly stronger and stiffer in females than males. We conclude that the congruent design of the TMJ, thin disk, biochemical content, and mechanical properties all reflect a structure-function relationship within the TMJ disk that is likely designed for the sea otter's hard diet and continuous food intake.