Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 µm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (µSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis.

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 µm non-motile cocci, S. aureus deforms smaller than 0.5 µm in the sub-micron channels of the OLCN. Here we present the µSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The µSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the µSiM-CA. However the µSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.

What Regions of the Distal Clavicle Have the Greatest Bone Mineral Density and Cortical Thickness? A Cadaveric Study.

BACKGROUND: Osteosynthesis of distal clavicle fractures can be challenging because of comminution, poor bone quality, and deforming forces at the fracture site. A better understanding of regional differences in the bone structure of the distal clavicle is critical to refine fracture fixation strategies, but the variations in BMD and cortical thickness throughout the distal clavicle have not been previously described. PURPOSE: /questions (1) Which distal clavicular regions have the greatest BMD? (2) Which distal clavicular regions have the greatest cortical thickness values? METHODS: Ten distal clavicle specimens were dissected from cadaveric shoulders. Eight specimens were female and two were male, with a mean (range) age of 63 years (59 to 67). The specimens were selected to match known epidemiology, as distal clavicular fractures occur more commonly in older patients with osteoporotic bone, and clavicular fractures in older patients are more common in females than males. The clavicles were then imaged using quantitative micro-CT to create 3-D images. The BMD and cortical thickness were calculated for 10 regions of interest in each specimen. These regions were selected to represent locations where distal clavicular fractures commonly occur and locations of likely bony comminution. Findings were compared between different regions using repeated measures ANOVA with Geiser-Greenhouse correction, followed by Bonferroni method multiple comparison testing. Effect size was also calculated to estimate the magnitude of difference between regions. RESULTS: The four most medial regions of the distal clavicle contained the greatest BMD (anterior intertubercle space 887 ± 31 mgHA/cc, posterior intertubercle space 879 ± 26 mgHA/cc, anterior conoid tubercle 900 ± 21 mgHA/cc, posterior conoid tubercle 896 ± 27 mgHA/cc), while the four most lateral regions contained the least BMD (anterior lateral distal clavicle 804 ± 32 mgHA/cc, posterior lateral distal clavicle 800 ± 38 mgHA/cc, anterior medial distal clavicle 815 ± 27 mgHA/cc, posterior medial distal clavicle 795 ± 26 mgHA/cc). All four most medial regions had greater BMD than the four most lateral regions, with p < 0.001 for all comparisons. For the BMD ANOVA, η was determined to be 0.81, representing a large effect size. The four most medial regions of the distal clavicle also had the greatest cortical thickness (anterior intertubercle space 0.7 ± 0.2 mm, posterior intertubercle space 0.7 ± 0.3 mm, anterior conoid tubercle 0.9 ± 0.2 mm, posterior conoid tubercle 0.7 ± 0.2 mm), while the four most lateral regions had the smallest cortical thickness (anterior lateral distal clavicle 0.2 ± 0.1 mm, posterior lateral distal clavicle 0.2 ± 0.1 mm, anterior medial distal clavicle 0.3 ± 0.1 mm, posterior medial distal clavicle 0.2 ± 0.1 mm). All four most medial regions had greater cortical thickness than the four most lateral regions, with p < 0.001 for all comparisons. For the cortical thickness ANOVA, η was determined to be 0.80, representing a large effect size. No differences in BMDs and cortical thicknesses were found between anterior and posterior regions of interest in any given area. CONCLUSIONS: In the distal clavicle, BMD and cortical thickness are greatest in the conoid tubercle and intertubercle space. When compared with clavicular regions lateral to the trapezoid tubercle, the BMD and cortical thickness of the conoid tubercle and intertubercle space were increased, with a large magnitude of difference. CLINICAL RELEVANCE: Distal clavicular fractures are prone to comminution and modern treatment strategies have centered on the use of locking plate technology and/or suspensory fixation between the coracoid and the clavicle. However, screw pullout or cortical button pull through are known complications of locking plate and suspensory fixation, respectively. Therefore, it seems intuitive that implant placement during internal fixation of distal clavicle fractures should take advantage of the best-available bone. Although osteosynthesis was not directly studied, our study suggests that the best screw purchase in the distal clavicle is available in the areas of the conoid tubercle and intertubercle space, as these areas had the best bone quality. Targeting these areas during implant fixation would likely reduce implant failure and strengthen fixation. Future studies should build on our findings to determine if osteosynthesis of distal clavicular fractures with targeted screw purchase or cortical button placement in the conoid tubercle and intertubercle space increase fixation strength and decreases construct failure. Furthermore, our findings provide consideration for novel distal clavicular locking plate designs with modified screw trajectories or refined surgical techniques with suspensory fixation implants to reliably capture these areas of greatest bone quality.

Reconstruction of the Superior Glenoid Labrum With Biceps Tendon Autograft: A Cadaveric Biomechanical Study.

PURPOSE: To describe 2 superior labral reconstruction techniques using long head of the biceps (LHB) autograft and to investigate the ability of the 2 reconstruction techniques to restore superior restraint to the glenohumeral joint compared with superior labrum-deficient models. METHODS: In this biomechanical study, 10 cadaveric shoulders were cycled on a servohydraulic machine while the force required to cause superior subluxation was recorded. Each specimen was cycled under 4 conditions: intact labrum, SLAP tear, posterior (9- to 12-o'clock position) labral reconstruction using LHB autograft (superior labral reconstruction 1 [SLR1]), and 180° (9- to 3-o'clock position) labral reconstruction using LHB autograft (superior labral reconstruction 2 [SLR2]). RESULTS: The mean peak force required to cause superior subluxation in the intact labrum was 32.75 N versus 19.75 N in the SLAP tear (P = .0120). SLR1 required a mean peak force of 31.23 N versus 44.09 N for SLR2 (P = .0175). SLR1 required 94.96% of the force needed in the intact labrum to cause subluxation, whereas SLR2 required 140.6%. SLR1 and SLR2 required 34.21% higher (P = .0074) and 79.84% higher (P = .0033) forces, respectively, to generate subluxation compared with the SLAP tear state. CONCLUSIONS: Both proposed superior labral reconstruction techniques increased the force needed for humeral head superior migration in the setting of a labral tear. SLR1 (posterior labral reconstruction) closely matched the constraint of an intact labrum, whereas SLR2 (180° labral reconstruction) provided greater superior constraint than an intact labrum. CLINICAL RELEVANCE: The natural history of irreparable rotator cuff tears results in superior glenohumeral escape and eventual arthrosis. The superior glenoid labrum is an important contributor to superior glenohumeral constraint and is often degenerated in this setting. Clinical application of the 2 described superior labral reconstruction techniques may improve glenohumeral superior stability in patients with rotator cuff disease and superior labral deficiency.

Diblock Copolymer Hydrophobicity Facilitates Efficient Gene Silencing and Cytocompatible Nanoparticle-Mediated siRNA Delivery to Musculoskeletal Cell Types.

pH-responsive diblock copolymers provide tailorable nanoparticle (NP) architecture and chemistry critical for siRNA delivery. Here, diblock polymers varying in first (corona) and second (core) block molecular weight (Mn), corona/core ratio, and core hydrophobicity (%BMA) were synthesized to determine their effect on siRNA delivery in murine tenocytes (mTenocyte) and murine and human mesenchymal stem cells (mMSC and hMSCs, respectively). NP-mediated siRNA uptake, gene silencing, and cytocompatibility were quantified. Uptake is positively correlated with first block Mn in mTenocytes and hMSCs (p ≤ 0.0005). All NP resulted in significant gene silencing that was positively correlated with %BMA (p < 0.05) in all cell types. Cytocompatibility was reduced in mTenocytes compared to MSCs (p < 0.0001). %BMA was positively correlated with cytocompatibility in MSCs (p < 0.05), suggesting stable NP are more cytocompatible. Overall, this study shows that NP-siRNA cytocompatibility is cell type dependent, and hydrophobicity (%BMA) is the critical diblock copolymer property for efficient gene silencing in musculoskeletal cell types.

TGF-ß1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair.

Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, Transforming Growth Factor Beta 1(TGF-ß1), but the effects of TGF-ß1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-ß1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-ß1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-ß1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-ß1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-ß1 on protease activity were completely abolished in tendon cells from homozygous plasminogen activator inhibitor 1 (PAI-1) knockout (KO) mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-ß1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries.

The Effect of the Epitendinous Suture on Gliding in a Cadaveric Model of Zone II Flexor Tendon Repair.

PURPOSE: We hypothesized that increasing core sutures (4-6) may be preferable in terms of gliding coefficient (GC) measurements when compared with adding an epitendinous suture to zone II flexor tendon repairs. We hypothesized that the inclusion of epitendinous suture in 2 standard repairs would contribute negatively to the GC of the repaired tendon. METHODS: Nineteen fresh-frozen cadaveric fingers were used for testing. We compared a control group (dissected digits without repair) and 4-strand or 6-strand core tendon repairs with and without epitendinous suture. Arc of motion was driven by direct loading, and digital images were acquired and analyzed. Outcomes were defined as the difference in GC between the native uninjured and the repaired state at each load. A linear mixed-model analysis was performed with comparisons between repairs to evaluate the statistically relevant differences between groups. RESULTS: The test of fixed effects in the linear model revealed that repair type and the use of epitendinous suture significantly affected the change in GC. The addition of an epitendinous suture produced a significant decrement in gliding regardless of repair type. CONCLUSIONS: There was significant improvement in GC with the omission of the epitendinous suture in both repair types (4- or 6-strand). CLINICAL RELEVANCE: The epitendinous suture used in this model resulted in poorer gliding of the repair, which may correspond with an expected increase in catching or triggering.

Flexor digitorum superficialis repair outside the A2 pulley after zone II laceration: gliding and bowstringing.

PURPOSE: To evaluate the changes in maximum flexion angle, gliding coefficient, and bowstringing after a combined repair of both flexor tendons with the flexor digitorum superficialis (FDS) rerouted outside the A2 pulley in cadaveric hands. METHODS: We performed 4 different repairs on cadaveric hands, with each repair tested on 9 unique digits. In total, 12 cadaveric hands and 36 digits were used. The thumb and little finger were removed from each hand and excluded from testing. Group 1 was sham surgery. Group 2 combined flexor digitorum profundus (FDP) and FDS laceration and repair with both slips of the FDS repaired inside the A2 pulley. Group 3 was FDP repair with one slip of the FDS repaired inside A2 and the other slip left unrepaired. Group 4 was FDP repair with both slips of the FDS rerouted and repaired outside the A2 pulley. Maximum flexion angle, gliding coefficient, and bowstringing were measured in simulated active digital motion for each group. RESULTS: Rerouting and repairing the FDS outside the A2 pulley (group 4) significantly lowered gliding coefficient compared with repairs with both slips inside A2, with values similar to sham surgery. We observed no significant differences in maximum flexion angle among the 4 groups. Increased bowstringing was observed with both slips of the FDS repaired and rerouted outside the A2 pulley. CONCLUSIONS: In this cadaveric model, repair of both slips of the FDS outside the A2 pulley improved the gliding coefficient relative to repair within the A2 pulley, which suggests decreased resistance to finger flexion. Repair of the FDS outside the A2 pulley led to a slight increase in bowstringing of the FDS tendon. CLINICAL RELEVANCE: We describe a technique for managing combined laceration of the FDP and FDS tendons that improves gliding function and merits consideration.

Biomechanical Effects of Subtalar Joint Fusion and Medial Ligament Reconstruction in Simulated Progressive Collapsing Foot Deformity.

BACKGROUND: The purpose of this study is to investigate the biomechanical effect of medial displacement calcaneal osteotomy (MDCO), subtalar joint fusion (SF), and medial ligament reconstruction (MLR: deltoid-spring ligament) in a severe flatfoot model. We hypothesized that (1) combination of MDCO and SF improves the tibiotalar and foot alignment in severe progressive collapsing foot deformity (PCFD) cadaver model. (2) However, if a residual valgus heel alignment remains after MCDO and SF, it can lead to increased medial ligament strain, foot malalignment, and tibiotalar valgus tilt, which will be mitigated by the addition of MLR. METHODS: Ten fresh-frozen cadaveric foot specimens were used to create a severe flatfoot model. The foot alignment changes, including the talo-first metatarsal angle in the axial and sagittal planes, subtalar angle, and tibiotalar angle in the coronal plane, were measured. The angles were measured at the initial condition, after creating the severe flatfoot model, and after each successive reconstructive procedure in the following order: (1) MDCO, (2) SF, and (3) MLR. RESULTS: Tibiotalar valgus tilt was decreased with the MDCO procedure: 4.4 vs 1.0 degrees (P = .04). Adding in situ SF to the MDCO led to increased tibiotalar tilt to 2.5 degrees was different from the initial condition (P = .01). Although the tibiotalar valgus tilt was significantly decreased after adding the MLR to the MDCO/SF procedure compared with the severe flatfoot model (0.8 vs 4.4 degrees, P = .03), no significant difference in the tibiotalar valgus tilt was observed between MDCO/SF and MDCO/SF with MLR. CONCLUSION: Our results demonstrated that MDCO significantly improved forefoot abduction and medial arch alignment, with no significant additional improvement observed with addition of SF. Following SF, a residual valgus heel alignment can contribute to subsequent tibiotalar valgus tilt. The addition of MLR did not show significantly decreased tibiotalar valgus tilt following SF. CLINICAL RELEVANCE: Residual valgus heel alignment after subtalar joint fusion in the surgical treatment of PCFD can lead to increased medial ligament strain. Although MLR might be considered for providing medial stability, it may not necessarily prevent the development of tibiotalar valgus tilt.

High-throughput micro-CT analysis identifies sex-dependent biomarkers of erosive arthritis in TNF-Tg mice and differential response to anti-TNF therapy.

BACKGROUND: Development of reliable disease activity biomarkers is critical for diagnostics, prognostics, and novel drug development. Although computed tomography (CT) is the gold-standard for quantification of bone erosions, there are no consensus approaches or rationales for utilization of specific outcome measures of erosive arthritis in complex joints. In the case of preclinical models, such as sexually dimorphic tumor necrosis factor transgenic (TNF-Tg) mice, disease severity is routinely quantified in the ankle through manual segmentation of the talus or small regions of adjacent bones primarily due to the ease in measurement. Herein, we sought to determine the particular hindpaw bones that represent reliable biomarkers of sex-dependent disease progression to guide future investigation and analysis. METHODS: Hindpaw micro-CT was performed on wild-type (n = 4 male, n = 4 female) and TNF-Tg (n = 4 male, n = 7 female) mice at monthly intervals from 2-5 (females) and 2-8-months (males) of age, since female TNF-Tg mice exhibit early mortality from cardiopulmonary disease at approximately 5-6-months. Further, 8-month-old WT (n = 4) and TNF-Tg males treated with anti-TNF monoclonal antibodies (n = 5) or IgG placebo isotype controls (n = 6) for 6-weeks were imaged with micro-CT every 3-weeks. For image analysis, we utilized our recently developed high-throughput and semi-automated segmentation strategy in Amira software. Synovial and osteoclast histology of ankle joints was quantified using Visiopharm. RESULTS: First, we demonstrated that the accuracy of automated segmentation, determined through analysis of ~9000 individual bones by a single user, was comparable in wild-type and TNF-Tg hindpaws before correction (79.2±8.9% vs 80.1±5.1%, p = 0.52). Compared to other bone compartments, the tarsal region demonstrated a sudden, specific, and significant bone volume reduction in female TNF-Tg mice, but not in males, by 5-months (4-months 4.3± 0.22 vs 5-months 3.4± 0.62 mm3, p<0.05). Specifically, the cuboid showed significantly reduced bone volumes at early timepoints compared to other tarsals (i.e., 4-months: Cuboid -24.1±7.2% vs Talus -9.0±5.9% of 2-month baseline). Additional bones localized to the anterolateral region of the ankle also exhibited dramatic erosions in the tarsal region of females, coinciding with increased synovitis and osteoclasts. In TNF-Tg male mice with severe arthritis, the talus and calcaneus exhibited the most sensitive response to anti-TNF therapy measured by effect size of bone volume change over treatment period. CONCLUSIONS: We demonstrated that sexually dimorphic changes in arthritic hindpaws of TNF-Tg mice are bone-specific, where the cuboid serves as a reliable early biomarker of erosive arthritis in female mice. Adoption of automated segmentation approaches in pre-clinical or clinical models has potential to translate quantitative biomarkers to monitor bone erosions in disease and evaluate therapeutic efficacy.

Engineering superficial zone features in tissue engineered cartilage.

A major challenge in cartilage tissue engineering is the need to recreate the native tissue's anisotropic extracellular matrix structure. This anisotropy has important mechanical and biological consequences and could be crucial for integrative repair. Here, we report that hydrodynamic conditions that mimic the motion-induced flow fields in between the articular surfaces in the synovial joint induce the formation of a distinct superficial layer in tissue engineered cartilage hydrogels, with enhanced production of cartilage matrix proteoglycan and Type II collagen. Moreover, the flow stimulation at the surface induces the production of the surface zone protein Proteoglycan 4 (aka PRG4 or lubricin). Analysis of second harmonic generation signature of collagen in this superficial layer reveals a highly aligned fibrillar matrix that resembles the alignment pattern in native tissue's surface zone, suggesting that mimicking synovial fluid flow at the cartilage surface in hydrodynamic bioreactors could be key to creating engineered cartilage with superficial zone features.

Cellular and molecular factors in flexor tendon repair and adhesions: a histological and gene expression analysis.

Flexor tendon healing is mediated by cell proliferation, migration, and extracellular matrix synthesis that contribute to the formation of scar tissue and adhesion. The biological mechanisms of flexor tendon adhesion formation have been linked to transforming growth factor ß (TGF-ß). To elucidate the cellular and molecular events in this pathology, we implanted live flexor digitorum longus grafts from the reporter mouse Rosa26(LacZ/+) in wild-type recipients, and used histological ß-galactosidase (ß-gal) staining to evaluate the intrinsic versus extrinsic cellular origins of scar, and reverse transcription-polymerase chain reaction to measure gene expression of TGF-ß and its receptors, extracellular matrix proteins, and matrix metalloproteinases (MMPs) and their regulators. Over the course of healing, graft cellularity and ß-gal activity progressively increased, and ß-gal-positive cells migrated out of the Rosa26(LacZ/+) graft. In addition, there was an evidence of influx of host cells (ß-gal-negative) into the gliding space and the graft, suggesting that both graft and host cells contribute to adhesions. Interestingly, we observed a biphasic pattern in which Tgfb1 expression was the highest in the early phases of healing and gradually decreased thereafter, whereas Tgfb3 increased and remained upregulated later. The expression of TGF-ß receptors was also upregulated throughout the healing phases. In addition, type III collagen and fibronectin were upregulated during the proliferative phase of healing, confirming that murine flexor tendon heals by scar tissue. Furthermore, gene expression of MMPs showed a differential pattern in which inflammatory MMPs were the highest early and matrix MMPs increased over time. These findings offer important insights into the complex cellular and molecular factors during flexor tendon healing.

Mechanisms of bone fragility in a mouse model of glucocorticoid-treated rheumatoid arthritis: implications for insufficiency fracture risk.

OBJECTIVE: Glucocorticoid (GC) therapy is associated with increased risk of fracture in patients with rheumatoid arthritis (RA). To elucidate the cause of this increased risk, we examined the effects of chronic erosive inflammatory arthritis and GC treatment on bone quality, structure, and biomechanical properties in a murine model. METHODS: Mice with established arthritis and expressing human tumor necrosis factor α (TNFα) transgene (Tg) and their wild-type (WT) littermates were continually treated with GC (prednisolone 5 mg/kg/day via subcutaneous controlled-release pellet) or placebo for 14, 28, or 42 days. Microstructure, biomechanical properties, chemical composition, and morphology of the tibiae and lumbar vertebral bodies were assessed by micro-computed tomography, biomechanical testing, Raman spectroscopy, and histology, respectively. Serum markers of bone turnover were also determined. RESULTS: TNF-Tg and GC treatment additively decreased mechanical strength and stiffness in both the tibiae and the vertebral bodies. GC treatment in the TNF-Tg mice increased the ductility of tibiae under torsional loading. These changes were associated with significant alterations in the biochemical and structural composition of the mineral and organic components of the bone matrix, a decrease in osteoblast activity and bone formation, and an increase in osteoclast activity. CONCLUSION: Our findings indicate that the concomitant decrease in bone strength and increase in bone ductility associated with chronic inflammation and GC therapy, coupled with the significant changes in the bone quality and structure, may increase the susceptibility of the bone to failure under low-energy loading. This may explain the mechanism of symptomatic insufficiency fractures in patients with RA receiving GC therapy who do not have radiographic manifestations of fracture.

Addition of a suture anchor for coracoclavicular fixation to a superior locking plate improves stability of type IIB distal clavicle fractures.

PURPOSE: The purpose of this study was to determine the effect of coracoclavicular (CC) fixation on biomechanical stability in type IIB distal clavicle fractures fixed with plate and screws. METHODS: Twelve fresh-frozen matched cadaveric specimens were used to create type IIB distal clavicle fractures. Dual-energy x-ray absorptiometry (DEXA) scans ensured similar bone quality. Group 1 (6 specimens) was stabilized with a superior precontoured distal clavicle locking plate and supplemental suture anchor CC fixation. Group 2 (6 specimens) followed the same construct without CC fixation. Each specimen was cyclically loaded in the coronal plane at 40 to 80 N for 17,500 cycles. Load-to-failure testing was performed on the specimens that did not fail cyclic loading. Outcome measures included mode of failure and the number of cycles or load required to create 10 mm of displacement in the construct. RESULTS: All specimens (12 of 12) completed cyclic testing without failure and underwent load-to-failure testing. Group 1 specimens failed at a mean of 808.5 N (range, 635.4 to 952.3 N), whereas group 2 specimens failed at a mean of 401.3 N (range, 283.6 to 656.0 N) (P = .005). Group 1 specimens failed by anchor pullout without coracoid fracture (4 of 6) and distal clavicle fracture fragment fragmentation (1 of 6); one specimen did not fail at the maximal load the materials testing machine was capable of exerting (1,000 N). Group 2 specimens failed by distal clavicle fracture fragment fragmentation (3 of 6) and acromioclavicular (AC) joint displacement (1 of 6); 2 specimens did not fail at the maximal load of the materials testing machine. CONCLUSIONS: During cyclic loading, type IIB distal clavicle fractures with and without CC fixation remain stable. CC fixation adds stability to type IIB distal clavicle fractures fixed with plate and screws when loaded to failure. CLINICAL RELEVANCE: CC fixation for distal clavicle fractures is a useful adjunct to plate-and-screw fixation to augment stability of the fracture.

Comparison of an all-inside suture technique with traditional pull-out suture and suture anchor repair techniques for flexor digitorum profundus attachment to bone.

PURPOSE: One goal in repairing zone 1 flexor digitorum profundus (FDP) injuries is to create a tendon-bone construct strong enough to allow early rehabilitation while minimizing morbidity. This study compares an all-inside suture repair technique biomechanically with pull-out suture and double-suture anchor repairs. METHODS: Repairs were performed on 30 cadaver fingers. In all-inside suture repairs (n = 8), the FDP tendon was attached to bone with two 3-0 Ethibond sutures and tied over the dorsal aspect of distal phalanx. Pull-out suture repairs (n = 8) were performed with 2-0 Prolene suture and tied over a dorsal button. There were 2 suture anchor repair groups: Arthrex Micro Corkscrew anchors preloaded with 2-0 FiberWire suture (n = 7) and Depuy Micro Mitek anchors preloaded with 3-0 Orthocord suture (n = 7). Repair constructs were tested using a servohydraulic materials testing system and loaded until the repair lost 75% of its strength. RESULTS: There were no statistically significant differences in tensile stiffness, ultimate load, or work to failure between the repairs. Failure mode was suture stretch and gap formation greater than 2 mm at the repair site for all pull-out suture repairs and for 7 of 8 all-inside suture repairs. Two of the Arthrex Micro Corkscrew repairs and 5 of the Depuy Micro Mitek repairs failed by anchor pull-out. CONCLUSIONS: This cadaveric biomechanical study showed no difference in tensile stiffness, ultimate load, and work to failures between an all-inside suture repair technique for zone 1 FDP repairs and previously described pull-out suture and suture anchor repair techniques. The all-inside suture technique also has the advantages of avoiding an external button and the cost of anchors. Therefore, it should be considered as an alternative to other techniques. CLINICAL RELEVANCE: This study introduces a new FDP reattachment technique that avoids some of the shortcomings of current techniques.

Structural and biomechanical responses of osseous healing: a novel murine nonunion model.

BACKGROUND: Understanding the biological mechanisms of why certain fractures are at risk for delayed healing or nonunion requires translational animal models that take advantage of transgenic and other genetic manipulation technologies. Reliable murine nonunion models can be an important tool to understand the biology of nonunion. In this study, we report the results of a recently established model for creating critical defects that lead to atrophic nonunions based on a unique fracture fixation technique. MATERIALS AND METHODS: Subcritical (0.6 mm long) and critical (1.6 mm long) defects were created in femurs of 10-week-old double transgenic (Col1/Col2) mice and stabilized using a custom-designed plate and four screws. Four groups were used: normal, sham, subcritical, and critical. Histology (n = 3 for each group) was analyzed at 2 and 5 weeks, and micro-computed tomography (µCT) and torsional biomechanics (n = 12 for each group) were analyzed at 5 weeks. RESULTS: Subcritical defects showed healing at 2 weeks and were completely healed by 5 weeks, with biomechanical properties not significantly different from normal controls. However, critical defects showed no healing by histology or µCT. These nonunion fractures also displayed no torsional stiffness or strength in 10 of 12 cases. CONCLUSIONS: Our murine fracture model creates reproducible and reliable nonunions and can serve as an ideal platform for studying molecular pathways to contrast healing versus nonhealing events and for evaluating innovative therapeutic approaches to promote healing of a challenging osseous injury.

Detection of osteoporotic-related bone changes and prediction of distal radius strength using Raman spectra from excised human cadaver finger bones.

While osteoporosis is reliably diagnosed using dual energy X-ray absorptiometry (DXA), screening rates are alarmingly low, contributing to preventable fractures. Raman spectroscopy (RS) can detect biochemical changes that occur in bones transcutaneously and can arguably be more accessible than DXA as a fracture risk assessment. A reasonable approach to translate RS is to interrogate phalangeal bones of human hands, where the soft tissues covering the bone are less likely to hamper transcutaneous measurements. To that end, we set out to first determine whether Raman spectra obtained from phalangeal bones correlate with distal radius fracture strength, which can predict subsequent osteoporotic fractures at the spine and hip. We performed RS upon diaphyseal and epiphyseal regions of exposed proximal phalanges from 12 cadaver forearms classified as healthy (n = 3), osteopenic (n = 4), or osteoporotic (n = 5) based on wrist T-scores measured by DXA. We observed a significant decrease in phosphate to matrix ratio and a significant increase in carbonate substitution in the osteoporotic phalanges relative to healthy and osteopenic phalanges. Multivariate regression models produced wrist T-score estimates with significant correlation to the DXA-measured values (r = 0.79). Furthermore, by accounting for phalangeal RS parameters, body mass index, and age, a multivariate regression significantly predicted distal radius strength measured in a simulated-fall biomechanical test (r = 0.81). These findings demonstrate the feasibility of interrogating the phalanges using RS for bone quality assessment of distant clinical sites of fragility fractures, such as the wrist. Future work will address transcutaneous measurement challenges as another requirement for scale-up and translation.

Effects of Acetabular Screws on the Initial Stability of Porous Coated Acetabular Implants in Revision Total Hip Arthroplasty.

INTRODUCTION: Revision total hip arthroplasty in the setting of acetabular bone loss remains a challenging clinical entity. Deficiencies of the acetabular rim, walls, and/or columns may limit the bony surface area and initial acetabular construct stability necessary for osseointegration of cementless components. Press-fit acetabular components with supplemental acetabular screw fixation represent a common technique aimed to minimize implant micromotion and allow for definitive osseointegration. Although acetabular screw fixation is commonly practiced in revision hip arthroplasty, few studies to date have examined the screw properties associated with maximal acetabular construct stability. The purpose of the present report is to examine acetabular screw fixation in a pelvis model mimicking Paprosky IIB acetabular bone loss. METHODS: Measuring bone-implant interface micromotion as a surrogate for initial implant stability, experimental models assessed the effect of screw number, screw length, and screw position on construct stability subject to a cyclic loading protocol designed to replicate joint reaction forces of two common daily activities. RESULTS: Trends towards increasing stability were demonstrated with increasing screw number, increasing screw length, and concentrating screws in the supra-acetabular dome. All experimental constructs yielded micromotion levels sufficient for bone ingrowth, except when screws in the dome were moved to the pubis and ischium. CONCLUSIONS: When using a porous coated revision acetabular implant to treat Paprosky IIB defects, screws should be used, and furthermore, increasing number, length, and position within the acetabular dome may help further stabilize the construct.

Human tendon-on-a-chip for modeling vascular inflammatory fibrosis.

Understanding vascular inflammation and myofibroblast crosstalk is critical to developing therapies for fibrotic diseases. Here we report the development of a novel human Tendon-on-a-Chip (hToC) to model this crosstalk in peritendinous adhesions, a debilitating fibrotic condition affecting flexor tendon, which currently lacks biological therapies. The hToC enables cellular and paracrine interactions between a vascular compartment harboring endothelial cells and monocytes with a tissue hydrogel compartment containing tendon fibroblasts and macrophages. We find that the hToC replicates in vivo inflammatory and fibrotic phenotypes in preclinical and clinical samples, including myofibroblast differentiation and tissue contraction, excessive ECM deposition, and inflammatory cytokines secretion. We further show that the fibrotic phenotypes are driven by the transmigration of monocytes from the vascular to the tissue compartments of the chip. We demonstrate significant overlap in fibrotic transcriptional signatures in the hToC with human tenolysis samples, including mTOR signaling, a regulatory nexus of fibrosis across various organs. Treatment with rapamycin suppressed the fibrotic phenotype on the hToC, which validates the hToC as a preclinical alternative for investigating fibrosis and testing therapeutics.

Role of the Mitochondrial Permeability Transition in Bone Metabolism and Aging.

The mitochondrial permeability transition pore (MPTP) and its positive regulator, cyclophilin D (CypD), play important pathophysiological roles in aging. In bone tissue, higher CypD expression and pore activity are found in aging; however, a causal relationship between CypD/MPTP and bone degeneration needs to be established. We previously reported that CypD expression and MPTP activity are downregulated during osteoblast (OB) differentiation and that manipulations in CypD expression affect OB differentiation and function. Using a newly developed OB-specific CypD/MPTP gain-of-function (GOF) mouse model, we here present evidence that overexpression of a constitutively active K166Q mutant of CypD (caCypD) impairs OB energy metabolism and function, and bone morphological and biomechanical parameters. Specifically, in a spatial-dependent and sex-dependent manner, OB-specific CypD GOF led to a decrease in oxidative phosphorylation (OxPhos) levels, higher oxidative stress, and general metabolic adaptations coincident with the decreased bone organic matrix content in long bones. Interestingly, accelerated bone degeneration was present in vertebral bones regardless of sex. Overall, our work confirms CypD/MPTP overactivation as an important pathophysiological mechanism leading to bone degeneration and fragility in aging. © 2023 American Society for Bone and Mineral Research (ASBMR).