CAMKK2 is upregulated in primary human osteoarthritis and its inhibition protects against chondrocyte apoptosis.

OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) in human osteoarthritis. MATERIALS AND METHODS: Paired osteochondral plugs and articular chondrocytes were isolated from the relatively healthier (intact) and damaged portions of human femoral heads collected from patients undergoing total hip arthroplasty for primary osteoarthritis (OA). Cartilage from femoral plugs were either flash frozen for gene expression analysis or histology and immunohistochemistry. Chondrocyte apoptosis in the presence or absence of CAMKK2 inhibition was measured using flow cytometry. CAMKK2 overexpression and knockdown in articular chondrocytes were achieved via Lentivirus- and siRNA-mediated approaches respectively, and their effect on pro-apoptotic and cartilage catabolic mechanisms was assessed by immunoblotting. RESULTS: CAMKK2 mRNA and protein levels were elevated in articular chondrocytes from human OA cartilage compared to paired healthier intact samples. This increase was associated with elevated catabolic marker matrix metalloproteinase 13 (MMP-13), and diminished anabolic markers aggrecan (ACAN) and type II collagen (COL2A1) levels. OA chondrocytes displayed enhanced apoptosis, which was suppressed following pharmacological inhibition of CAMKK2. Levels of MMP13, pSTAT3, and the pro-apoptotic marker BAX became elevated when CAMKK2, but not its kinase-defective mutant was overexpressed, whereas knockdown of the kinase decreased the levels of these proteins. CONCLUSIONS: CAMKK2 is upregulated in human OA cartilage and is associated with elevated levels of pro-apoptotic and catabolic proteins. Inhibition or knockdown of CAMKK2 led to decreased chondrocyte apoptosis and catabolic protein levels, whereas its overexpression elevated them. CAMKK2 may be a therapeutic target to prevent or mitigate human OA.

A conceptual time window-based model for the early stratification of trauma patients.

Progress in the testing of therapies targeting the immune response following trauma, a leading cause of morbidity and mortality worldwide, has been slow. We propose that the design of interventional trials in trauma would benefit from a scheme or platform that could support the identification and implementation of prognostic strategies for patient stratification. Here, we propose a stratification scheme based on defined time periods or windows following the traumatic event. This 'time-window' model allows for the incorporation of prognostic variables ranging from circulating biomarkers and clinical data to patient-specific information such as gene variants to predict adverse short- or long-term outcomes. A number of circulating biomarkers, including cell injury markers and damage-associated molecular patterns (DAMPs), and inflammatory mediators have been shown to correlate with adverse outcomes after trauma. Likewise, several single nucleotide polymorphisms (SNPs) associate with complications or death in trauma patients. This review summarizes the status of our understanding of the prognostic value of these classes of variables in predicting outcomes in trauma patients. Strategies for the incorporation of these prognostic variables into schemes designed to stratify trauma patients, such as our time-window model, are also discussed.

Severe muscle trauma triggers heightened and prolonged local musculoskeletal inflammation and impairs adjacent tibia fracture healing.

OBJECTIVES: Complicated fracture healing is often associated with the severity of surrounding muscle tissue trauma. Since inflammation is a primary determinant of musculoskeletal health and regeneration, it is plausible that delayed healing and non-unions are partly caused by compounding local inflammation in response to concomitant muscle trauma. METHODS AND RESULTS: To investigate this possibility, a Lewis rat open fracture model [tibia osteotomy with adjacent tibialis anterior (TA) muscle volumetric muscle loss (VML) injury] was interrogated. We observed that VML injury impaired tibia healing, as indicated by diminished mechanical strength and decreased mineralized bone within the fracture callus, as well as continued presence of cartilage instead of woven bone 28 days post-injury. The VML injured muscle presented innate and adaptive immune responses that were atypical of canonical muscle injury healing. Additionally, the VML injury resulted in a perturbation of the inflammatory phase of fracture healing, as indicated by elevations of CD3(+) lymphocytes and CD68+ macrophages in the fracture callus at 3 and 14d post-injury, respectively. CONCLUSIONS: These data indicate that heightened and sustained innate and adaptive immune responses to traumatized muscle are associated with impaired fracture healing and may be targeted for the prevention of delayed and non-union following musculoskeletal trauma.

Muscle-bone interactions during fracture healing.

Although it is generally accepted that the rate and strength of fracture healing is intimately linked to the integrity of surrounding soft tissues, the contribution of muscle has largely been viewed as a vascular supply for oxygen and nutrient exchange. However, more is becoming known about the cellular and paracrine contributions of muscle to the fracture healing process. Research has shown that muscle is capable of supplying osteoprogenitor cells in cases where the periosteum is insufficient, and the muscular osteoprogenitors possess similar osteogenic potential to those derived from the periosteum. Muscle's secrotome includes proteins capable of inhibiting or enhancing osteogenesis and myogenesis following musculoskeletal injury and can be garnered for therapeutic use in patients with traumatic musculoskeletal injuries. In this review, we will highlight the current knowledge on muscle-bone interaction in the context of fracture healing as well as concisely present the current models to study such interactions.

Strain-dependent oxidant release in articular cartilage originates from mitochondria.

Mechanical loading is essential for articular cartilage homeostasis and plays a central role in the cartilage pathology, yet the mechanotransduction processes that underlie these effects remain unclear. Previously, we showed that lethal amounts of reactive oxygen species (ROS) were liberated from the mitochondria in response to mechanical insult and that chondrocyte deformation may be a source of ROS. To this end, we hypothesized that mechanically induced mitochondrial ROS is related to the magnitude of cartilage deformation. To test this, we measured axial tissue strains in cartilage explants subjected to semi-confined compressive stresses of 0, 0.05, 0.1, 0.25, 0.5, or 1.0 MPa. The presence of ROS was then determined by confocal imaging with dihydroethidium, an oxidant sensitive fluorescent probe. Our results indicated that ROS levels increased linearly relative to the magnitude of axial strains (r(2) = 0.87, p < 0.05), and significant cell death was observed at strains >40%. By contrast, hydrostatic stress, which causes minimal tissue strain, had no significant effect. Cell-permeable superoxide dismutase mimetic Mn(III)tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride significantly decreased ROS levels at 0.5 and 0.25 MPa. Electron transport chain inhibitor, rotenone, and cytoskeletal inhibitor, cytochalasin B, significantly decreased ROS levels at 0.25 MPa. Our findings strongly suggest that ROS and mitochondrial oxidants contribute to cartilage mechanobiology.

Mitochondrial electron transport and glycolysis are coupled in articular cartilage.

OBJECTIVE: Although the majority of the adenosine triphosphate (ATP) in chondrocytes is made by glycolysis rather than by oxidative phosphorylation in mitochondria there is evidence to suggest that reactive oxygen species produced by mitochondrial electron transport (ET) help to maintain cellular redox balance in favor of glycolysis. The objective of this study was to test this hypothesis by determining if rotenone, which inhibits ET and blocks oxidant production inhibits glycolytic ATP synthesis. DESIGN: Bovine osteochondral explants were treated with rotenone, an ET inhibitor; or oligomycin an ATP synthase inhibitor; or 2-fluoro-2-deoxy-D-glucose, a glycolysis inhibiter; or peroxide, an exogenous oxidant; or mitoquinone (MitoQ), a mitochondria-targeted anti-oxidant. Cartilage extracts were assayed for ATP, nicotine adenine dinucleotide (NAD+/H), and culture medium was assayed for pyruvate and lactate after 24 h of treatment. Imaging studies were used to measure superoxide production in cartilage. RESULTS: Rotenone and 2-FG caused a significant decline in cartilage ATP (P < 0.001). In contrast, ATP levels were not affected by oligomycin. Peroxide treatment blocked rotenone effects on ATP, while treatment with MitoQ significantly suppressed ATP levels. Rotenone and 2-FG caused a significant decline in pyruvate, but not in lactate production. NADH:NAD+ ratios decreased significantly in both rotenone and 2-FG-treated explants (P < 0.05). Rotenone also significantly reduced superoxide production. CONCLUSIONS: These findings showing a link between glycolysis and ET are consistent with previous reports on the critical need for oxidants to support normal chondrocyte metabolism. They suggest a novel role for mitochondria in cartilage homeostasis that is independent of oxidative phosphorylation.

The effect of incongruity and instability on contact stress directional gradients in human cadaveric ankles.

OBJECTIVE: Measure incongruity and instability-associated changes in transient contact stress directional gradients in a human cadaveric ankle model. METHODS: Seven cadaveric ankles were subjected to quasi-physiologic forces and motion under intact conditions and with a stepoff incongruity of the anterior one-third of the distal tibia. Anterior/posterior forces were modulated to create incongruous specimens that either maintained a stable articulation between the talus and distal tibia or developed gross instability during motion. Real-time contact stresses were measured using a custom-designed ankle stress transducer at 132 Hz. Contact stress data were differentiated using a central-differencing formula to calculate transient contact stress directional gradients over the entire ankle articulation. RESULTS: Transient 95th percentile contact stress directional gradient values increased by 30 and 100%, respectively, in stable-incongruous and unstable-incongruous conditions compared to intact conditions. Compared to stable-incongruous conditions, transient contact stress directional gradients increased by 60% in unstable-incongruous conditions. CONCLUSIONS: Instability resulted in greater percentage increases in transient contact stress directional gradients compared to incongruity. Pathologic increases in contact stress directional gradients potentially play an important role in the etiology of post-traumatic arthritis.

Stance-phase aggregate contact stress and contact stress gradient changes resulting from articular surface stepoffs in human cadaveric ankles.

OBJECTIVE: Determine how stepoff incongruities of the distal tibia affect aggregate (whole-cycle) contact stresses and contact stress gradients for a complete motion cycle in human cadaveric ankles. METHOD: Ten human cadaveric ankles were subjected to quasiphysiologic forces during stance-phase range of motion. Each specimen was loaded intact, with anatomic reduction of the anterolateral quarter of the distal tibia, and with increasing stepoffs of the anterolateral fragment up to 4.0mm. Transient contact stresses were measured using a custom-built, real-time stress transducer that sampled stresses at 132Hz at 1472 separate foci (sensels). Aggregate stresses were calculated by summing the sequential transient stress values multiplied by the transient sampling duration for the complete motion cycle at each sensel. Transient contact stress gradients were calculated at each sensel using a central-differencing formula applied to adjacent transient stress measurements. Aggregate contact stress gradients were calculated by vector summation of sequential transient stress gradients multiplied by the sampling duration. RESULTS: Compared to the intact configuration, anatomic reduction of the fragment caused minimal changes in aggregate contact stresses and stress gradients (30% increase compared to intact values). In contrast, stepoffs caused substantial increases (200% increase compared to intact values) in peak and mean whole-cycle stresses and gradients. CONCLUSIONS: Aggregate contact stresses and stress gradients quantify loading history for the complete motion cycle. Incongruity-associated changes in aggregate stresses and gradients are a surrogate for "accumulated" damage over a motion cycle in stepoff specimens. These loading abnormalities may be important determinants of posttraumatic arthritis.

Trabecular bone strain changes associated with cartilage defects in the proximal and distal tibia.

Intraarticular fractures with cartilage defects can lead to post-traumatic arthritis (PTA). The purpose of this study was to determine how cartilage defects affect load transmission through subchondral trabecular bone in human cadaveric knees and ankles to further understand the pathomechanics of PTA. We created full-thickness cartilage defects in the meniscectomized proximal tibia and distal tibia and measured changes in trabecular bone strain using Texture Correlation. Texture Correlation compares high quality digital images made from contact radiographs of unloaded samples to images of the same sample under load to measure trabecular bone strain. Cartilage defects caused trabecular bone strain to decrease in the proximal tibia and increase in the distal tibia. The column of bone directly beneath the defect in the tibial plateau had the most significant reduction in strain. In the distal tibia, strain near the jointline and in the anterior third had the most significant increases in strain. The distal tibia had greater strain changes with small defects. The clinical course of intraarticular fractures of the proximal and distal tibia are markedly different. We postulate that disturbances in load transmission through the subchondral bone caused by cartilage defects may be important mechanical determinants of PTA.

Characteristics of pedicle screw loading. Effect of sagittal insertion angle on intrapedicular bending moments.

STUDY DESIGN: A bending analysis of pedicle screws inserted into vertebral body analogues. Intravertebral and intrapedicular pedicle screw bending moments were studied as a function of sagittal insertion angle. OBJECTIVES: To determine how the pedicle screw bending moment is affected by changes in the insertion angle. SUMMARY OF BACKGROUND DATA: There is a significant incidence of failure when pedicle screws are used to instrument unstable spinal segments. Extrinsic factors that affect screw bending failure have been poorly characterized. Previous work has demonstrated that intrapedicular pedicle screw bending moments are significantly affected by the sagittal location and depth of pedicle screw placement. METHODS: Pedicle screw transducers were inserted in analogue vertebrae at one of three orientations: 7 degrees cephalad (toward the superior endplate), 7 degrees caudal (toward the inferior endplate), or parallel to the superior endplate (control). An axial load was applied to the superior endplate of the vertebra, and screw bending moments were recorded directly from the transducers. RESULTS: Screws angled 7 degrees cephalad developed significantly greater mean intrapedicular bending moments compared with screws inserted caudal or control screws. There was no significant difference in bending moments realized within the vertebral body for the three screw positions. CONCLUSIONS: Angulating pedicle screws toward the superior endplate increased bending moments within the pedicle. If attention to optimal screw insertion technique can reduce bending moments and potential for screw failure without increasing morbidity, surgical risk, or operative time, then proper insertion technique takes on new importance.

Characteristics of pedicle screw loading. Effect of surgical technique on intravertebral and intrapedicular bending moments.

STUDY DESIGN: A static nondestructive bending analysis of pedicle screws inserted into vertebral analogues was conducted. Pedicle screw load was studied as a function of variables in insertion technique. OBJECTIVES: To determine how the sagittal bending moment in pedicle screws is affected by changes in pedicle screw length, insertional depth, and sagittal placement. BACKGROUND DATA: An unexpectedly high rate of clinical failure has been observed in pedicle screws used in short-segment instrumentation for unstable burst fractures. The majority of screws fail in sagittal bending within the pedicle. Little is known of the insertion technical factors that affect in situ loads incurred by pedicle screws. METHODS: Synthetic vertebral analogues were fabricated. Pedicle screws internally instrumented with strain gauges were used as load transducers to determine screw bending moments within the pedicle and body of the analogue. Analogues were loaded in compression to simulate loading of an unstable burst fracture. RESULTS: Screw bending moments within the pedicle increased 33% and 52% when screws were left 3 mm and 5 mm short of full insertion. Intrapedicular moments increased 20% to 29% in screws inserted superiorly or inferiorly within the pedicle. Thirty-five-millimeter screws developed intrapedicular moments 16% higher than 40-mm and 45-mm screws. CONCLUSIONS: In situ pedicle screw loads increased significantly as a direct result of variations in surgical technique. Screws left short of full insertion, placed off center in the sagittal plane of the pedicle, or less than 40 mm long developed increased intrapedicular bending moments.

The effect of bone quality on pedicle screw loading in axial instability. A synthetic model.

STUDY DESIGN: In this biomechanical analysis of pedicle screw bending moments, custom-fabricated vertebral analogues were loaded in axial compression to produce sagittal bending forces. Moments were measured directly from internally instrumented pedicle screws. OBJECTIVES: To establish the role of cancellous vertebral modulus on pedicle screw bending moments within the vertebral body and the vertebral pedicle. SUMMARY OF BACKGROUND DATA: Pedicle screws are often used to manage axial instability of the spine. Clinical studies report a high incidence of screw bending failure, resulting in kyphosis and pain in some patients. Factors predisposing to bending failure are not well understood, although recent studies have shown that vertebral morphometry is important. METHODS: Axially canullated 7.0-mm pedicle screws, internally instrumented with paired strain gauges, were inserted into analogue vertebrae of uniform dimension. Cancellous modulus was varied from 25-100 MPa. Screws were rigidly mounted to a vertical testing frame, and axial loads were applied to the superior vertebral endplate, producing sagittal bending moments. Moments were recorded from gauges applied in the intrapedicular and intravertebral portions of the screw. Mean moments were compared using a Student's t test, with significance defined as P < 0.05. RESULTS: Cancellous modulus did not affect bending moments experienced in either the intrapedicular or intravertebral portions of the pedicle screws. Gauge accuracy was excellent, and with no gauge drift. CONCLUSIONS: Although small changes in pedicle morphometry can alter screw bending moments significantly, changes in cancellous modulus had no measurable impact on bending moments at these same loads. Bone density is likely to play a limited role in screw bending failure.

The effect of pedicle morphometry on pedicle screw loading. A synthetic model.

STUDY DESIGN: Static nondestructive bending analysis of pedicle screws inserted into vertebral analogues was conducted. Pedicle screw bending load was studied as a function of pedicle morphometry. OBJECTIVES: To determine how sagittal bending moment in pedicle screws is affected by changes in pedicle height, length, and width. BACKGROUND DATA: An unexpectedly high rate of clinical failure has been observed in pedicle screws used in short-segment instrumentation for axially unstable fractures. The majority of screws fail in sagittal bending within the pedicle. To date, little is known of the exogenous factors that affect in situ loads incurred by pedicle screws. METHODS: Synthetic vertebral analogues were fabricated, varying pedicle height, length, or width independently. Pedicle screws internally instrumented with strain gages were used as load transducers to determine screw bending moments within the pedicle and body of the analogue. Analogues were loaded in compression to simulate loading of an unstable burst fracture. RESULTS: Screw bending moments within the pedicle increased incrementally with increasing pedicle length, rising 30% as length increased from 8.0 mm to 12.0 mm. Screw moment increased 20% when pedicle height dropped below 15.0 mm, consistent with a threshold effect. Changes in pedicle width did not affect screw loads within the pedicle. CONCLUSIONS: In situ pedicle screw loads increased significantly as pedicle length increased and as pedicle height decreased. Pedicle screws instrumented internally with strain gages are an effective research instrument allowing measurement of in situ loading along the axis of the screw.

A device for the measurement of pedicle screw moments by means of internal strain gauges.

Pedicle screws are commonly used in spinal reconstruction, and failure of pedicle screws due to bending is a significant clinical problem. To measure the moments typically placed on pedicle screws in situ we instrumented 7 mm Cotrel-Dubousset (CD) pedicle screws with internally mounted strain gauges. The screws were designed to measure flexion-extension moments at a single cross-section as dictated by strain gauge placement. It is possible to measure moments of up to 12 Nm at any location along the length of the screw by constructing transducers with varying strain gauge placements. These transducers are capable of measuring moments at points located within the vertebra including the pedicle, which is where failure usually occurs clinically. Transducer output was both linear and reproducible. These transducers are being used to investigate the load transfer characteristics between the pedicle screw and the vertebra. This technique could be applied to investigations of load sharing in reconstruction plates, lag-screws, and cross-locked intramedullary nails.