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.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

PAI-1 mediates TGF-ß1-induced myofibroblast activation in tenocytes via mTOR signaling.

Transforming growth factor-beta (TGF-ß1) induces plasminogen activator inhibitor 1 (PAI-1) to effect fibrotic pathologies in several organs including tendon. Recent data implicated PAI-1 with inhibition of phosphatase and tensin homolog (PTEN) suggesting that PAI-1-induced adhesions involves phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (mTOR) signaling. Ergo, we investigated effects of TGF-ß1, PAI-1, and mTOR signaling crosstalk on myofibroblast activation, senescence, and proliferation in primary flexor tenocytes from wild-type (WT) and PAI-1 knockout (KO) mice. PAI-1 deletion blunted TGF-ß1-induced myofibroblast activation in murine flexor tenocytes and increased the gene expression of Mmp-2 to confer protective effects against fibrosis. While TGF-ß1 significantly reduced phosphorylation of PTEN in WT cells, PAI-1 deletion rescued the activation of PTEN. Despite that, there were no differences in TGF-ß1-induced activation of mTOR signaling (AKT, 4EBP1, and P70S6K) in WT or KO tenocytes. Phenotypic changes in distinct populations of WT or KO tenocytes exhibiting high or low mTOR activity were then examined. TGF-ß1 increased alpha-smooth muscle actin abundance in WT cells exhibiting high mTOR activity, but this increase was blunted in KO cells exhibiting high 4EBP1 activity but not in cells exhibiting high S6 activity. DNA damage (γH2AX) was increased with TGF-ß1 treatment in WT tenocytes but was blunted in KO cells exhibiting high mTOR activity. Increased mTOR activity enhanced proliferation (Ki67) in both WT and KO tenocytes. These findings point to a complex nexus of TGF-ß1, PAI-1, and mTOR signaling in regulating proliferation, myofibroblast differentiation, and senescence in tenocytes, which could define therapeutic targets for chronic tendon adhesions and other fibrotic pathologies.

Sourcing cells for in vitro models of human vascular barriers of inflammation.

The vascular system plays a critical role in the progression and resolution of inflammation. The contributions of the vascular endothelium to these processes, however, vary with tissue and disease state. Recently, tissue chip models have emerged as promising tools to understand human disease and for the development of personalized medicine approaches. Inclusion of a vascular component within these platforms is critical for properly evaluating most diseases, but many models to date use "generic" endothelial cells, which can preclude the identification of biomedically meaningful pathways and mechanisms. As the knowledge of vascular heterogeneity and immune cell trafficking throughout the body advances, tissue chip models should also advance to incorporate tissue-specific cells where possible. Here, we discuss the known heterogeneity of leukocyte trafficking in vascular beds of some commonly modeled tissues. We comment on the availability of different tissue-specific cell sources for endothelial cells and pericytes, with a focus on stem cell sources for the full realization of personalized medicine. We discuss sources available for the immune cells needed to model inflammatory processes and the findings of tissue chip models that have used the cells to studying transmigration.

Transcriptional regulation of cyclophilin D by BMP/Smad signaling and its role in osteogenic differentiation.

Cyclophilin D (CypD) promotes opening of the mitochondrial permeability transition pore (MPTP) which plays a key role in both cell physiology and pathology. It is, therefore, beneficial for cells to tightly regulate CypD and MPTP but little is known about such regulation. We have reported before that CypD is downregulated and MPTP deactivated during differentiation in various tissues. Herein, we identify BMP/Smad signaling, a major driver of differentiation, as a transcriptional regulator of the CypD gene, Ppif. Using osteogenic induction of mesenchymal lineage cells as a BMP/Smad activation-dependent differentiation model, we show that CypD is in fact transcriptionally repressed during this process. The importance of such CypD downregulation is evidenced by the negative effect of CypD 'rescue' via gain-of-function on osteogenesis both in vitro and in a mouse model. In sum, we characterized BMP/Smad signaling as a regulator of CypD expression and elucidated the role of CypD downregulation during cell differentiation.

Human Organ-on-a-Chip Microphysiological Systems to Model Musculoskeletal Pathologies and Accelerate Therapeutic Discovery.

Human Microphysiological Systems (hMPS), otherwise known as organ- and tissue-on-a-chip models, are an emerging technology with the potential to replace in vivo animal studies with in vitro models that emulate human physiology at basic levels. hMPS platforms are designed to overcome limitations of two-dimensional (2D) cell culture systems by mimicking 3D tissue organization and microenvironmental cues that are physiologically and clinically relevant. Unlike animal studies, hMPS models can be configured for high content or high throughput screening in preclinical drug development. Applications in modeling acute and chronic injuries in the musculoskeletal system are slowly developing. However, the complexity and load bearing nature of musculoskeletal tissues and joints present unique challenges related to our limited understanding of disease mechanisms and the lack of consensus biomarkers to guide biological therapy development. With emphasis on examples of modeling musculoskeletal tissues, joints on chips, and organoids, this review highlights current trends of microphysiological systems technology. The review surveys state-of-the-art design and fabrication considerations inspired by lessons from bioreactors and biological variables emphasizing the role of induced pluripotent stem cells and genetic engineering in creating isogenic, patient-specific multicellular hMPS. The major challenges in modeling musculoskeletal tissues using hMPS chips are identified, including incorporating biological barriers, simulating joint compartments and heterogenous tissue interfaces, simulating immune interactions and inflammatory factors, simulating effects of in vivo loading, recording nociceptors responses as surrogates for pain outcomes, modeling the dynamic injury and healing responses by monitoring secreted proteins in real time, and creating arrayed formats for robotic high throughput screens. Overcoming these barriers will revolutionize musculoskeletal research by enabling physiologically relevant, predictive models of human tissues and joint diseases to accelerate and de-risk therapeutic discovery and translation to the clinic.

A high-throughput semi-automated bone segmentation workflow for murine hindpaw micro-CT datasets.

INTRODUCTION: Micro-computed tomography (µCT) is a valuable imaging modality for longitudinal quantification of bone volumes to identify disease or treatment effects for a broad range of conditions that affect bone health. Complex structures, such as the hindpaw with up to 31 distinct bones in mice, have considerable analytic potential, but quantification is often limited to a single bone volume metric due to the intensive effort of manual segmentation. Herein, we introduce a high-throughput, user-friendly, and semi-automated method for segmentation of murine hindpaw µCT datasets. METHODS: In vivo µCT was performed on male (n = 4; 2-8-months) and female (n = 4; 2-5-months) C57BL/6 mice longitudinally each month. Additional 9.5-month-old male C57BL/6 hindpaws (n = 6 hindpaws) were imaged by ex vivo µCT to investigate the effects of resolution and integration time on analysis outcomes. The DICOMs were exported to Amira software for the watershed-based segmentation, and watershed markers were generated automatically at approximately 80% accuracy before user correction. The semi-automated segmentation method utilizes the original data, binary mask, and bone-specific markers that expand to the full volume of the bone using watershed algorithms. RESULTS: Compared to the conventional manual segmentation using Scanco software, the semi-automated approach produced similar raw bone volumes. The semi-automated segmentation also demonstrated a significant reduction in segmentation time for both experienced and novice users compared to standard manual segmentation. ICCs between experienced and novice users were >0.9 (excellent reliability) for all but 4 bones. DISCUSSION: The described semi-automated segmentation approach provides remarkable reliability and throughput advantages. Adoption of the semi-automated segmentation approach will provide standardization and reliability of bone volume measures across experienced and novice users and between institutions. The application of this model provides a considerable strategic advantage to accelerate various research opportunities in pre-clinical bone and joint analysis towards clinical translation.

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse.

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth's Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. RNA-seq analysis revealed that differentially expressed genes in the C57 healing tendon at 7 days post injury were functionally linked to fibrosis, immune system signaling and extracellular matrix (ECM) organization, while the differentially expressed genes in the MRL injured tendon were dominated by cell cycle pathways. These gene expression changes were associated with increased α-SMA+ myofibroblast and F4/80+ macrophage activation and abundant BCL-2 expression in the C57 injured tendons. Transcriptional analysis of upstream regulators using Ingenuity Pathway Analysis showed positive enrichment of TGFB1 in both C57 and MRL healing tendons, but with different downstream transcriptional effects. MRL tendons exhibited of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and fibrotic (ECM organization) pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair in MRL mice, and could give cues to improved tendon healing.

Cantilever Bending of Murine Femoral Necks.

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20° relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6° ± 1.5°, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis.

Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (µSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

Chondral Damage After Arthroscopic Repair Techniques for Acute Bony Bankart Lesions: A Biomechanical Study.

BACKGROUND: Bony Bankart lesions can be encountered during treatment of shoulder instability. Current arthroscopic bony Bankart repair techniques involve intra-articular suture placement, but the effect of these repair techniques on the integrity of the humeral head articular surface warrants further investigation. PURPOSE: To quantify the degree of humeral head articular cartilage damage secondary to current arthroscopic bony Bankart repair techniques in a cadaveric model. STUDY DESIGN: Controlled laboratory study. METHODS: Testing was performed in 13 matched pairs of cadaveric glenoids with simulated bony Bankart fractures, with a defect width of 25% of the glenoid diameter. Half of the fractures were repaired with a double-row technique, while the contralateral glenoids were repaired with a single-row technique. Samples were subjected to 20,000 cycles of internal-external rotation across a 90° arc at 2 Hz after a compressive load of 750 N, or 90% body weight (whichever was less) was applied to simulate wear. Cartilage defects on the humeral head were quantified through a custom MATLAB script. Mean cartilage cutout differences were analyzed by the Wilcoxon rank-sum test. RESULTS: Both single- and double-row repairs showed macroscopic damage. The histomorphometric analysis demonstrated that the double-row technique resulted in a significantly (P = .036) more chondral damage (mean, 57,489.1 µm2; SD, 61,262.2 µm2) than the single-row repair (mean, 28,763.5 µm2; SD, 24,4990.2 µm2). CONCLUSION: Both single-row and double-row arthroscopic bony Bankart fixation techniques resulted in damage to the humeral head articular cartilage in the concavity-compression model utilized in this study. The double-row fixation technique resulted in a significantly increased cutout to the humeral head cartilage after simulated wear in this cadaveric model. CLINICAL RELEVANCE: This study provides data demonstrating that placement of intra-articular suture during arthroscopic bony Bankart repair techniques may harm the humeral head cartilage. While the double-row repair of bony Bankart lesions is more stable, it results in increased cartilage damage. These findings suggest that alternative, cartilage-sparing arthroscopic techniques for bony Bankart repair should be investigated.

A Biomechanical, Cadaveric Evaluation of Single- Versus Double-Row Repair Techniques on Stability of Bony Bankart Lesions.

BACKGROUND: Previous studies comparing stability between single- and double-row arthroscopic bony Bankart repair techniques focused only on the measurements of tensile forces on the bony fragment without re-creating a more physiologic testing environment. PURPOSE: To compare dynamic stability and displacement between single- and double-row arthroscopic repair techniques for acute bony Bankart lesions in a concavity-compression cadaveric model simulating physiologic conditions. STUDY DESIGN: Controlled laboratory study. METHODS: Testing was performed on 13 matched pairs of cadaveric glenoids with simulated bony Bankart fractures with a defect width of 25% of the inferior glenoid diameter. Half of the fractures were repaired with a double-row technique, and the contralateral glenoids were repaired with a single-row technique. To determine dynamic biomechanical stability and ultimate step-off of the repairs, a 150-N load and 2000 cycles of internal-external rotation at 1 Hz were applied to specimens to simulate early rehabilitation. Toggle was quantified throughout cycling with a coordinate measuring machine. Three-dimensional spatial measurements were calculated. After cyclic loading, the fracture displacement was measured. RESULTS: The bony Bankart fragment-glenoid initial step-off was found to be significantly greater (P < .001) for the single-row technique (mean, 896 µm; SD, 282 µm) compared with the double-row technique (mean, 436 µm; SD, 313 µm). The motion toggle was found to be significantly greater (P = .017) for the single-row technique (mean, 994 µm; SD, 711 µm) compared with the double-row technique (mean, 408 µm; SD, 384 µm). The ultimate interface displacement was found to be significantly greater (P = .029) for the single-row technique (mean, 1265 µm; SD, 606 µm) compared with the double-row technique (mean, 795 µm; SD, 398 µm). CONCLUSION: Using a concavity-compression glenohumeral cadaveric model, we found that the double-row arthroscopic fixation technique for bony Bankart repair resulted in superior stability and decreased displacement during simulated rehabilitation when compared with the single-row repair technique. CLINICAL RELEVANCE: The findings from this study may help guide surgical decision-making by demonstrating superior biomechanical properties (improved initial step-off, motion toggle, and interface displacement) of the double-row bony Bankart repair technique when compared with single-row fixation. The double-row repair construct demonstrated increased stability of the bony Bankart fragment, which may improve bony Bankart healing.

Improved prediction of femoral fracture toughness in mice by combining standard medical imaging with Raman spectroscopy.

Bone fragility and fracture risk are assessed by measuring the areal bone mineral density (aBMD) using dual-energy X-ray absorptiometry (DXA). While aBMD correlates with bone strength, it is a poor predictor of fragility fracture risk. Alternatively, fracture toughness assesses the bone's resistance to crack propagation and fracture, making it a suitable bone quality metric. Here, we explored how femoral midshaft measurements from DXA, micro-computed tomography (µCT), and Raman spectroscopy could predict fracture toughness. We hypothesized that ovariectomy (OVX) decreases aBMD and fracture toughness compared to controls and we can optimize a multivariate assessment of bone quality by combining results from X-ray and Raman spectroscopy. Female mice underwent an OVX (n = 5) or sham (n = 5) surgery at 3 months of age. Femurs were excised 3 months after ovariectomy and assessed with Raman spectroscopy, µCT, and DXA. Subsequently, a notch was created on the anterior side of the mid-diaphysis of the femurs. Three-point bending induced a controlled fracture that initiated at the notch. The OVX mice had a significantly lower aBMD, cortical thickness, and fracture toughness when compared to controls (p < 0.05). A leave one out cross-validated (LOOCV) partial least squares regression (PLSR) model based only on the combination of aBMD and cortical thickness showed no significant predictive correlations with fracture toughness, whereas a PLSR model based on principal components derived from the full Raman spectra yielded significant prediction (r2 = 0.71, p < 0.05). Further, the PLSR model was improved by incorporating aBMD, cortical thickness, and principal components from Raman spectra (r2 = 0.92, p < 0.001). This exploratory study demonstrates combining X-ray with Raman spectroscopy leads to a more accurate assessment of bone fracture toughness and could be a useful diagnostic tool for the assessment of fragility fracture risk.

Determination of best Raman spectroscopy spatial offsets for transcutaneous bone quality assessments in human hands.

Spatially offset Raman spectroscopy (SORS) is able to detect bone signal transcutaneously and could assist in predicting bone fracture risk. Criteria for optimal source-detector offsets for transcutaneous human measurements, however, are not well-established. Although larger offsets yield a higher percentage of bone signal, the absolute amount of bone signal decreases. Spectral unmixing into bone, adipose, and non-adipose components was employed to quantify changes in bone signal to noise ratio across a range of offsets, and optimal offsets for phalanx and metacarpal measurements were determined. The bone signal to noise ratio was maximized at offsets ranging from 4-6 mm.

Effects of tamoxifen on tendon homeostasis and healing: Considerations for the use of tamoxifen-inducible mouse models.

The use of tamoxifen-inducible models of Cre recombinase in the tendon field is rapidly expanding, resulting in an enhanced understanding of tendon homeostasis and healing. However, the effects of tamoxifen on the tendon are not well-defined, which is particularly problematic given that tamoxifen can have both profibrotic and antifibrotic effects in a tissue-specific manner. Therefore, in the present study, we examined the effects of tamoxifen on tendon homeostasis and healing in male and female C57Bl/6J mice. Tamoxifen-treated mice were compared to corn oil (vehicle)-treated mice. In the "washout" treatment regimen, mice were treated with tamoxifen or corn oil for 3 days beginning 1 week prior to undergoing complete transection and surgical repair of the flexor digitorum longus tendon. In the second regimen, mice were treated with tamoxifen or corn oil beginning on the day of surgery, daily through day 2 postsurgery, and every 48 hours thereafter (D0-2q48) until harvest. All repaired tendons and uninjured contralateral control tendons were harvested at day 14 postsurgery. Tamoxifen treatment had no effect on tendon healing in male mice, regardless of the treatment regimen, while Max load was significantly decreased in female repairs in the Tamoxifen washout group, relative to corn oil. In contrast, D0-2q48 corn oil treatment in female mice led to substantial disruptions in tendon homeostasis, relative to washout corn oil treatment. Collectively, these data clearly define the functional effects of tamoxifen and corn oil treatment in the tendon and inform future use of tamoxifen-inducible genetic models.

A Mouse Femoral Ostectomy Model to Assess Bone Graft Substitutes.

The shortcomings of autografts and allografts in bone defect healing have prompted researchers to develop suitable alternatives. Numerous biomaterials have been developed as bone graft substitutes each with their own advantages and disadvantages. However, in order to test if these biomaterials provide an adequate replacement of the clinical standard, a clinically representative animal model is needed to test their efficacy. In this chapter, we describe a mouse model that establishes a critical sized defect in the mid-diaphysis of the femur to evaluate the performance of bone graft substitutes. This is achieved by performing a femoral ostectomy and stabilization utilizing a femoral plate and titanium screws. The resulting defect enables the bone regenerative potential of bone graft substitutes to be investigated. Lastly, we provide instruction on assessing the torsional strength of the healed femurs to quantitatively evaluate the degree of healing as a primary outcome measure.

NF-κB activation persists into the remodeling phase of tendon healing and promotes myofibroblast survival.

Although inflammation is necessary during the early phases of tissue repair, persistent inflammation contributes to fibrosis. Acute tendon injuries often heal through a fibrotic mechanism, which impedes regeneration and functional recovery. Because inflammation mediated by nuclear factor κB (NF-κB) signaling is implicated in this process, we examined the spatial, temporal, and cell type-specific activation profile of canonical NF-κB signaling during tendon healing. NF-κB signaling was maintained through all phases of tendon healing in mice, including the remodeling phase, and tenocytes and myofibroblasts from the Scleraxis (Scx) lineage were the predominant populations that retained NF-κB activation into the late stages of repair. We confirmed persistent NF-κB activation in myofibroblasts in human tendon scar tissue. Deleting the canonical NF-κB kinase, IKKß, in Scx-lineage cells in mice increased apoptosis and the deposition of the matrix protein periostin during the late stages of tendon repair, suggesting that persistent NF-κB signaling may facilitate myofibroblast survival and fibrotic progression. Consistent with this, myofibroblasts in human tendon scar samples displayed enhanced prosurvival signaling compared to control tissue. Together, these data suggest that NF-κB may contribute to fibrotic tendon healing through both inflammation-dependent and inflammation-independent functions, such as NF-κB-mediated cell survival.

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.

A brief history of tendon and ligament bioreactors: Impact and future prospects.

Bioreactors are powerful tools with the potential to model tissue development and disease in vitro. For nearly four decades, bioreactors have been used to create tendon and ligament tissue-engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of two- and three-dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue-engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell-cell and cell-matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades.

Peritalar Kinematics With Combined Deltoid-Spring Ligament Reconstruction in Simulated Advanced Adult Acquired Flatfoot Deformity.

BACKGROUND: Adult acquired flatfoot deformity (AAFD) is a complex and progressive deformity involving the ligamentous structures of the medial peritalar joints. Recent anatomic studies demonstrated that the spring and deltoid ligaments form a greater medial ligament complex, the tibiocalcaneonavicular ligament (TCNL), which provides medial stability to the talonavicular, subtalar, and tibiotalar joints. The aim of this study was to assess the biomechanical effect of a spring ligament tear on the peritalar stability. The secondary aim was to assess the effect of TCNL reconstruction in restoration of peritalar stability in comparison with other medial stabilization procedures, anatomic spring or deltoid ligament reconstructions, in a cadaveric flatfoot model. METHODS: Ten fresh-frozen cadaveric foot specimens were used. Reflective markers were mounted on the tibia, talus, navicular, calcaneus, and first metatarsal. Peritalar joint kinematics were captured by a multiple-camera motion capture system. Mild, moderate, and severe flatfoot models were created by sequential sectioning of medial capsuloligament complex followed by cyclic axial loading. Spring only, deltoid only, and combined deltoid-spring ligament (TCNL) reconstructions were performed. The relative kinematic changes were compared using 2-way analysis of variance (ANOVA). RESULTS: Compared with the initial condition, we noted significantly increased valgus alignment of the subtalar joint of 5.1 ± 2.3 degrees (P = .031) and 5.8 ± 2.7 degrees (P < .01) with increased size of the spring ligament tear to create moderate to severe flatfoot, respectively. We noted an increased tibiotalar valgus angle of 5.1 ± 2.0 degrees (P = .03) in the severe model. Although all medial ligament reconstruction methods were able to correct forefoot abduction, the TCNL reconstruction was able to correct both the subtalar and tibiotalar valgus deformity (P = .04 and P = .02, respectively). CONCLUSION: The TCNL complex provided stability to the talonavicular, subtalar, and tibiotalar joints. The combined deltoid-spring ligament (TCNL) reconstructions restored peritalar kinematics better than isolated spring or deltoid ligament reconstruction in the severe AAFD model. CLINICAL RELEVANCE: The combined deltoid-spring ligament (TCNL) reconstruction maybe considered in advanced AAFD with medial peritalar instability: stage IIB with a large spring ligament tear or stage IV.