Douglas J. Adams

Tensile properties of the human femur-anterior cruciate ligament-tibia complex The effects of specimen age and orientation

The structural properties of 27 pairs of human cadaver knees were evaluated. Specimens were equally divided into three groups of nine pairs each based on age: younger (22 to 35 years), middle (40 to 50 years), and older (60 to 97 years). Anterior-posterior displacement tests with the intact knee at 30° and 90° of flexion revealed a significant effect of knee flexion angle, but not of specimen age. Tensile tests of the femur-ACL- tibia complex were performed at 30° of knee flexion with the ACL aligned vertically along the direction of applied tensile load. One knee from each pair was oriented anatomically (anatomical orientation), and the contralateral knee was oriented with the tibia aligned vertically (tibial orientation). Structural properties of the femur-ACL-tibia complex, as represented by the linear stiffness, ultimate load, and energy absorbed, were found to decrease significantly with specimen age and were also found to have higher values in specimens tested in the anatomical orientation. In the younger specimens, linear stiffness (242 ± 28 N/mm) and ulti mate load (2160 ± 157 N) values found when the femur- ACL-tibia complex was tested in the anatomical orien tation were higher than those reported previously in the literature. These values provide new baseline data for the design and selection of grafts for ACL replacement in an attempt to reproduce normal knee kinematics.

Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur

Although skeletal pain plays a major role in reducing the quality of life in patients suffering from osteoarthritis, Pagets disease, sickle cell anemia and bone cancer, little is known about the mechanisms that generate and maintain this pain. To define the peripheral fibers involved in transmitting and modulating skeletal pain, we used immunohistochemistry with antigen retrieval, confocal microscopy and three-dimensional image reconstruction of the bone to examine the sensory and sympathetic innervation of mineralized bone, bone marrow and periosteum of the normal mouse femur. Thinly myelinated and unmyelinated peptidergic sensory fibers were labeled with antibodies raised against calcitonin gene-related peptide (CGRP) and the unmyelinated, non-peptidergic sensory fibers were labeled with the isolectin B4 (Bandeira simplicifolia). Myelinated sensory fibers were labeled with an antibody raised against 200-kDa neurofilament H (clone RT-97). Sympathetic fibers were labeled with an antibody raised against tyrosine hydroxylase. CGRP, RT-97, and tyrosine hydroxylase immunoreactive fibers, but not isolectin B4 positive fibers, were present throughout the bone marrow, mineralized bone and the periosteum. While the periosteum is the most densely innervated tissue, when the total volume of each tissue is considered, the bone marrow receives the greatest total number of sensory and sympathetic fibers followed by mineralized bone and then periosteum. Understanding the sensory and sympathetic innervation of bone should provide a better understanding of the mechanisms that drive bone pain and aid in developing therapeutic strategies for treating skeletal pain.

Hyponatremia-induced osteoporosis

There is a high prevalence of chronic hyponatremia in the elderly, frequently owing to the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Recent reports have shown that even mild hyponatremia is associated with impaired gait stability and increased falls. An increased risk of falls among elderly hyponatremic patients represents a risk factor for fractures, which would be further amplified if hyponatremia also contributed metabolically to bone loss. To evaluate this possibility, we studied a rat model of SIADH and analyzed data from the Third National Health and Nutrition Examination Survey (NHANES III). In rats, dual‐energy X‐ray absorptiometry (DXA) analysis of excised femurs established that hyponatremia for 3 months significantly reduced bone mineral density by approximately 30% compared with normonatremic control rats. Moreover, micro‐computed tomography (µCT) and histomorphometric analyses indicated that hyponatremia markedly reduced both trabecular and cortical bone via increased bone resorption and decreased bone formation. Analysis of data from adults in NHANES III by linear regression models showed that mild hyponatremia is associated with increased odds of osteoporosis (T‐score –2.5 or less) at the hip [odds ratio (OR) = 2.85; 95% confidence interval (CI) 1.03–7.86; p < .01]; all models were adjusted for age, sex, race, body mass index (BMI), physical activity, history of diuretic use, history of smoking, and serum 25‐hydroxyvitamin D [25(OH)D] levels. Our results represent the first demonstration that chronic hyponatremia causes a substantial reduction of bone mass. Cross‐sectional human data showing that hyponatremia is associated with significantly increased odds of osteoporosis are consistent with the experimental data in rodents. Our combined results suggest that bone quality should be assessed in all patients with chronic hyponatremia.

Micro-CT enables microlocalisation and quantification of Her2-targeted gold nanoparticles within tumour regions

OBJECTIVES Gold nanoparticles are of interest as potential in vivo diagnostic and therapeutic agents, as X-ray contrast agents, drug delivery vehicles and radiation enhancers. The aim of this study was to quantitatively determine their targeting and microlocalisation in mouse tumour models after intravenous injection by using micro-CT. METHODS Gold nanoparticles (15 nm) were coated with polyethylene glycol and covalently coupled to anti-Her2 antibodies (Herceptin). In vitro, conjugates incubated with Her2+ (BT-474) and Her2- (MCF7) human breast cancer cells showed specific targeted binding with a Her2+ to Her2- gold ratio of 39.4±2.7:1. Nude mice, simultaneously bearing subcutaneous Her2+ and Her2- human breast tumours in opposite thighs were prepared. Gold nanoparticles alone, conjugated to Herceptin or to a non-specific antibody were compared. After intravenous injection of the gold nanoparticles, gold concentrations were determined by atomic absorption spectroscopy. Microlocalisation of gold was carried out by calibrated micro-CT, giving both the radiodensities and gold concentrations in tumour and non-tumour tissue. RESULTS All gold nanoparticle constructs showed accumulation, predominantly at tumour peripheries. However, the Herceptin-gold nanoparticles showed the best specific uptake in their periphery (15.8±1.7% injected dose per gram), 1.6-fold higher than Her2- tumours and 22-fold higher than surrounding muscle. Imaging readily enabled detection of small, 1.5 mm-thick tumours. CONCLUSION In this pre-clinical study, antibody-targeted 15 nm gold nanoparticles showed preferential uptake in cognate tumours, but even untargeted gold nanoparticles enhanced the visibility of tumour peripheries and enabled detection of millimetre-sized tumours. Micro-CT enabled quantification within various regions of a tumour.

The Effects of Knee Motion and External Loading on the Length of the Anterior Cruciate Ligament (ACL): A Kinematic Study

A six-degrees-of-freedom mechanical linkage device was designed and used to study the unconstrained motion of ten intact human cadaver knees. The knees were subjected to externally applied varus and valgus (V-V) moments up to 14 N-m as well as anterior and posterior (A-P) loads up to 100 N. Tests were done at four knee flexion angles; 0, 30, 45, and 90 deg. Significant coupled axial tibial rotation was found, up to 21.0 deg for V-V loading (at 90 deg of flexion) and 14.2 deg for A-P loading (at 45 deg of flexion). Subsequently, the knees were dissected and the locations of the insertion sites to the femur and tibia for the anteromedial (AM), posterolateral (PL), and intermediate (IM) portions of the ACL were identified. The distances between the insertion sites for all external loading conditions were calculated. In the case when the external load was zero, the AM portion of the ACL lengthened with knee flexion, while the PL portion shortened and the intermediate (IM) portion did not change in length. With the application of 14 N-m valgus moment, the PL and IM portions of the ACL lengthened significantly more than the AM portion (p less than 0.001). With the application of 100 N anterior load, the AM portion lengthened slightly less than the PL portion, which lengthened slightly less than the IM portion (p less than 0.005). In general, the amount of lengthening of the three portions of the ACL during valgus and anterior loading was observed to increase with knee flexion angle (p less than 0.001).

Medial Opening Wedge Tibial Osteotomy and the Sagittal Plane The Effect of Increasing Tibial Slope on Tibiofemoral Contact Pressure

Background Altering the tibial slope in an anterior cruciate ligament–deficient knee has been shown to affect anterior-posterior tibial translation. The effects on articular contact pressure of altering tibial slope during a high tibial osteotomy are unknown. Hypotheses Performing an opening wedge osteotomy anterior to the midaxial line will increase tibial slope. Increasing tibial slope with a high tibial osteotomy in an anterior cruciate ligament–deficient knee redistributes tibiofemoral joint contact pressures onto the posterior tibial plateau. Study Design Controlled laboratory study. Methods Medial opening wedge high tibial osteotomies were performed, and a plate fixation with a known diameter inset was placed along the medial tibia in an anterior position and a posterior position on 9 cadaveric knees. Medial and lateral tibiofemoral contact pressures were measured at the resulting 2 different tibial slopes in both ligament-intact and ligament-deficient states using thin electronic sensors. Results Anterior plate application resulted in an increase in posterior tibial slope by an average of 6.6°(P <.001) compared with posterior plate placement. After medial opening wedge high tibial osteotomy, the mean peak lateral tibiofemoral contact pressure (3.4 MPa) was significantly greater (P=.002) than was the mean peak medial pressure (2.6 MPa). In ligament-intact specimens, altering the tibial slope did not significantly shift peak contact pressures. However, in ligament-deficient knees, increasing tibial slope by an average of 5.5° significantly redistributed the location of peak intra-articular pressure, shifting it posteriorly by 24% (P=.003). Conclusion Increasing tibial slope in anterior cruciate ligament–deficient knees with a high tibial osteotomy redistributes pressure into the posterior tibial plateau. Clinical Relevance In knees with chronic anterior cruciate ligament deficiency, posteromedial compartment degeneration is observed. Inadvertent redistribution of contact pressure into this area may be a cause of pain and premature clinical failure after medial opening wedge tibial osteotomy.

Use of an alpha-smooth muscle actin GFP reporter to identify an osteoprogenitor population

Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of alpha-smooth muscle actin (alpha-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of alpha-SMA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of alpha-SMA+ cells towards osteoprogenitor lineage. Utilizing alpha-SMAGFP transgene, alpha-SMAGFP+ positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the alpha-SMAGFP+ population derived from bone marrow or adipose tissue. In adipose tissue, alpha-SMA+ cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, alpha-SMA+/CD45-/Sca1+ progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts.

Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In Vivo

We tested the hypothesis that induction of cyclo‐oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP‐2 induced COX‐2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP‐2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX‐2 promoter‐luciferase reporter construct (Pluc) and in MC3T3‐E1 cells transfected with Pluc. Deletion analysis identified the −300/−213‐bp region of the COX‐2 promoter as necessary for BMP‐2 stimulation of luciferase activity. Mutation of core‐binding factor activity 1 (muCbfa1) consensus sequence (5′‐AACCACA‐3′) at −267/−261 bp decreased BMP‐2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX‐2 knockout (−/−) and wild‐type (+/+) mice, the absence of COX‐2 expression reduced the BMP‐2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP‐2 induced osteoclast formation in cells from COX‐2+/+ mice but not in cells from COX‐2−/− mice. In vivo, BMP‐2 (10 μg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (μCT), was decreased by 78% in COX‐2−/− mice compared with COX‐2+/+ mice. We conclude that BMP‐2 transcriptionally induces COX‐2 in osteoblasts via a Cbfa1 binding site and that the BMP‐2 induction of COX‐2 can contribute to effects of BMP‐2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP‐2 stimulation of ectopic bone formation in vivo.

Ligamentous Lisfranc Joint Injuries: A Biomechanical Comparison of Dorsal Plate and Transarticular Screw Fixation

Background: The current treatment of displaced ligamentous injuries of the tarsometatarsal (TMT) joints is open reduction and rigid fixation using transarticular screws. This technique causes further articular surface damage that theoretically may increase the risk of arthritis. Should the screws break, hardware removal is difficult. An alternative method that avoids these potential complications is rigid fixation using dorsal plates. Methods: The displacement between the first metatarsal and medial cuneiform, the second metatarsal and intermediate cuneiform, the first and second metatarsal bases, and the medial cuneiform and second metatarsal base were measured in 10 matched pairs of fresh-frozen cadaver lower extremities in the unloaded and loaded condition. After sectioning the Lisfranc and TMT joint ligaments, measurements were repeated in the loaded condition. The first and second TMT joints of the right feet were fixed with transarticular 3.5-mm cortical screws while those of the left feet with were fixed with dorsal 2.7-mm 1/4 tubular plates. Measurements were then repeated in the unloaded and loaded condition. Results: After ligament sectioning, significantly increased first and second TMT joint subluxation with loading was seen. No significant difference was noted with direct comparison between plates and screws with respect to ability to realign the first and second TMT joints and to maintain TMT joint alignment during loading. The amount of articular surface destruction caused by one 3.5-mm screw was 2.0 ± 0.7% for the medial cuneiform, 2.6 ± 0.5% for the first metatarsal, 3.6 ± 1.2% for the intermediate cuneiform, and 3.6 ± 1.0% for the second metatarsal. Conclusions: The model reliably produced displacement of the first and second TMT joints consistent with a ligamentous Lisfranc injury. Transarticular screws and dorsal plates showed similar ability to reduce the first and second TMT joints after TMT and Lisfranc ligament transection and to resist TMT joint displacement with weightbearing load. Clinical relevance: Dorsal plating may be an alternative to transarticular screws in the treatment of displaced Lisfranc injuries.

The Effect of Triceps Surae Contracture Force on Plantar Foot Pressure Distribution

Background: Triceps surae contractures have been associated with foot and ankle pathology. Achilles tendon contractures have been shown to shift plantar foot pressure from the heel to the forefoot. The purpose of this study was to determine whether isolated gastrocnemius contractures had similar effects and to assess the effects of gastrocnemius or soleus contracture on midfoot plantar pressure. Methods: Ten fresh frozen cadaver below-knee specimens were loaded to 79 pounds (350N) plantar force with the foot unconstrained on a 10-degree dorsiflexed plate. Combinations of static gastrocnemius or soleus forces were applied in 3-lb increments and plantar pressure recordings were obtained for the hindfoot, midfoot, and forefoot regions. Results: The percentage of plantar force borne by the forefoot and midfoot increased with triceps surae force, while that borne by the hindfoot decreased (p ≤ 0.005). Increasing gastrocnemius force had similar results. Increasing triceps surae force from 0 to 21 lbs (93 N) increased average percent forefoot and midfoot force 59% and 38%, respectively, and reduced average percent hindfoot force 18%. Increasing gastrocnemius force from 0 to 18 lbs increased average percent forefoot and midfoot force 50% and 32%, respectively, and reduced average percent hindfoot force 16%. For a given triceps surae force, there was no statistical difference in pressure distribution noted between different combinations of gastrocnemius and soleus force. Conclusions: In a static model, increased triceps surae or isolated gastrocnemius force shifted weightbearing plantar pressure from the hindfoot to the midfoot and forefoot. Similar results were noted whether the triceps surae force was applied through the gastrocnemius or soleus or both. The results of this study are consistent with the clinical association of triceps surae contracture with foot and ankle disorders including diabetic foot ulcers and metatarsalgia. The similar effects with triceps surae force application through the gastrocnemius or soleus suggest that patients with isolated gastrocnemius contractures may obtain similar clinical benefits with potentially less morbidity after gastrocnemius aponeurosis lengthening as compared to Achilles tendon lengthening.