Long-Term Effects of Reproduction and Lactation on the Rat Supraspinatus Tendon and Proximal Humerus.

During pregnancy and breastfeeding, women undergo hormonal fluctuations required for fetal development, parturition, and infant growth. These changes have secondary consequences on the maternal musculoskeletal system, increasing the risk for joint pain and osteoporosis. Though hormone levels return to prepregnancy levels postpartum, women may experience lasting musculoskeletal pain. Sex disparities exist in the prevalence of musculoskeletal disorders, but it remains unclear how reproductive history may impact sex differences. Specifically, the effects of both reproductive history and sex on the rotator cuff have not been studied. Pregnancy and lactation affect bone microstructure, suggesting possible impairments at the enthesis of rotator cuff tendons, where tears commonly occur. Therefore, our objective was to evaluate how reproductive history affects sex differences of the supraspinatus tendon and proximal humerus using male, virgin female, and female rats with a history of reproduction (referred to as reproductive females). We hypothesized tendon mechanical properties and humeral bone microstructure would be inferior in reproductive females compared to virgin females. Results showed sex differences independent of reproductive history, including greater tendon midsubstance modulus but lower subchondral bone mineral density (BMD) in females. When considering reproductive history, reproductive rats exhibited reduced tendon insertion site modulus and trabecular bone micro-architecture compared to virgin females with no differences from males. Overall, our study identified long-term changes in supraspinatus tendon mechanical and humeral trabecular bone properties that result following pregnancy and lactation, highlighting the importance of considering reproductive history in investigations of sex differences in the physiology and pathology of rotator cuff injuries.

Short- and Long-Term Exercise Results in a Differential Achilles Tendon Mechanical Response.

The study was conducted to define the biomechanical response of rat Achilles tendon after a single bout of exercise and a short or long duration of daily exercise. We hypothesized that a single bout or a short duration of exercise would cause a transient decrease in Achilles tendon mechanical properties and a long duration of daily exercise would improve these properties. One hundred and thirty-six Sprague-Dawley rats were divided into cage activity (CA) or exercise (EX) groups for a single bout, short-term, or long-term exercise. Animals in single bout EX groups were euthanized, 3, 12, 24, or 48 h upon completion of a single bout of exercise (10 m/min, 1 h) on a flat treadmill. Animals in short-term EX groups ran on a flat treadmill for 3 days, 1, or 2 weeks while animals in long-term EX groups ran for 8 weeks. Tendon quasi-static and viscoelastic response was evaluated for all Achilles tendons. A single bout of exercise increased tendon stiffness after 48 h of recovery. Short-term exercise up to 1 week decreased cross-sectional area, stiffness, modulus, and dynamic modulus of the Achilles tendon. In contrast, 8 weeks of daily exercise increased stiffness, modulus, and dynamic modulus of the tendon. This study highlights the response of Achilles tendons to single and sustained bouts of exercise. Adequate time intervals are important to allow for tendon adaptations when initiating a new training regimen and overall beneficial effects to the Achilles tendon.

Induced Knockdown of Decorin, Alone and in Tandem With Biglycan Knockdown, Directly Increases Aged Murine Patellar Tendon Viscoelastic Properties.

Tendon injuries increase with age, yet the age-associated changes in tendon properties remain unexplained. Decorin and biglycan are two matrix proteoglycans that play complex roles in regulating tendon formation, maturation, and aging, most notably in extracellular matrix assembly and maintenance. However, the roles of decorin and biglycan have not been temporally isolated in a homeostatic aged context. The goal of this work was to temporally isolate and define the roles of decorin and biglycan in regulating aged murine patellar tendon mechanical properties. We hypothesized that decorin would have a larger influence than biglycan on aged tendon mechanical properties and that biglycan would have an additive role in this regulation. When decorin and biglycan were knocked down in aged tendons, minimal changes in gene expression were observed, implying that these models directly define the roles of decorin and biglycan in regulating tendon mechanical properties. Knockdown of decorin or biglycan led to minimal changes in quasi-static mechanical properties. However, decorin deficiency led to increases in stress relaxation and phase shift that were exacerbated when coupled with biglycan deficiency. This study highlights an important role for decorin, alone and in tandem with biglycan, in regulating aged tendon viscoelastic properties.

Pregnancy and Lactation Impair Subchondral Bone Leading to Reduced Rat Supraspinatus Tendon-to-Bone Insertion Site Failure Properties.

Pregnant women experience weight gain, gait changes, and biochemical fluctuations that impair joint function and alter the maternal skeleton. Hormonal changes increase pelvic ligament laxity in preparation for childbirth and affect peripheral joint laxity. Calcium demands also rise during pregnancy and lactation, resulting in reduced bone mineral density (BMD) and maternal bone loss. Altered tendon properties and bone loss during pregnancy and lactation may impact tendon insertion sites, such as rotator cuff tendons where insertion site ruptures are common. However, the effects of pregnancy and lactation at the tendon-to-bone interface have not been investigated. Therefore, the objective of this study was to evaluate supraspinatus tendon mechanical properties and insertion site microstructure during pregnancy, lactation, and postweaning recovery in female rats. We hypothesized that pregnancy and lactation would compromise supraspinatus tendon mechanical properties and subchondral bone microstructure. Female rats were divided into virgin, pregnancy, lactation, and recovery groups, and supraspinatus tendons were mechanically evaluated. Surprisingly, tendon mechanics was unaffected by pregnancy and lactation. However, tendon modulus decreased two-weeks postweaning. Additionally, tendons failed by bony avulsion at the insertion site, and the lactation group exhibited reduced failure properties corresponding to decreased subchondral bone mineralization. Lactation also resulted in dramatic bone loss at the epiphysis, but trabecular bone microarchitecture recovered postweaning. In conclusion, lactation following pregnancy impaired trabecular bone microstructure and subchondral bone mineralization, leading to reduced supraspinatus tendon-to-bone insertion site failure properties. These findings will contribute toward understanding the pathogenesis of tendon-to-bone disorders.

Tensile Mechanical Properties and Dynamic Collagen Fiber Re-Alignment of the Murine Cervix are Dramatically Altered Throughout Pregnancy.

The cervix is a unique organ able to dramatically change its shape and function by serving as a physical barrier for the growing fetus and then undergoing dramatic dilation allowing for delivery of a term infant. As a result, the cervix endures changing mechanical forces from the growing fetus. There is an emerging concept that the cervix may change or remodel "early" in many cases of spontaneous preterm birth (sPTB). However, the mechanical role of the cervix in both normal and preterm birth remains unclear. Therefore, the primary objective of this study was to determine the mechanical and structural responses of murine cervical tissue throughout a normal gestational time course. In this study, both tissue structural and material properties were determined via a quasi-static tensile load-to-failure test, while simultaneously obtaining dynamic collagen fiber re-alignment via cross-polarization imaging. This study demonstrated that the majority of the mechanical properties evaluated decreased at midgestation and not just at term, while collagen fiber re-alignment occurred earlier in the loading curve for cervices at term. This suggests that although structural changes in the cervix occur throughout gestation, the differences in material properties function in combination with collagen fiber re-alignment as mechanical precursors to regulate term gestation. This work lays a foundation for investigating cervical biomechanics and the role of the cervix in preterm birth.

Collagen V haploinsufficiency in female murine patellar tendons results in altered matrix engagement and cellular density, demonstrating decreased healing.

Collagen V (Col5) is a quantitatively minor component of collagen fibrils comprising tendon, however, plays a crucial role in regulation of development and dynamic healing processes. Clinically, patients with COL5a1 haploinsufficiency, known as classic Ehlers-Danlos Syndrome (cEDS), present with hyperextensible skin, joint instability and laxity, with females more likely to be affected. Previous studies in Col5-deficient mice indicated that reduced Col5a1 expression leads to a reduction in stiffness, fibril deposition, and altered fibril structure. Additionally, Col5-deficient male tendons demonstrated altered healing compared to wild-type tendons, however female mice have not yet been studied utilizing this model. Along with clinical differences between sexes in cEDS patient populations, differences in hormone physiology may be a factor influencing tendon health. Therefore, the objective of this study was to utilize a Col5a1+/ - female mouse model, to determine the effect of Col5 on tendon cell morphology, cell density, tissue composition, and mechanical properties throughout healing. We hypothesized that reduction in Col5 expression would result in an abnormal wound matrix post-injury, resulting in reduced mechanical properties compared to normal tendons. Following patellar tendon surgery, mice were euthanized at 1, 3, and 6-week post-injury. Col5-deficient tendons demonstrated altered and decreased healing compared to WT tendons. The lack of resolution in cellularity by 6-week post-injury in Col5-deficient tendons influenced the decreased mechanical properties. Stiffness did not increase post-injury in Col5-deficient mice, and collagen fiber realignment was delayed during mechanical loading. Therefore, increased Col5a1 expression post-injury is necessary to re-establish matrix engagement and cellularity throughout tendon healing.

Dose-dependent effects of enzyme replacement therapy on skeletal disease progression in mucopolysaccharidosis VII dogs.

Mucopolysaccharidosis (MPS) VII is an inherited lysosomal storage disorder characterized by deficient activity of the enzyme ß-glucuronidase. Skeletal abnormalities are common in patients and result in diminished quality of life. Enzyme replacement therapy (ERT) for MPS VII using recombinant human ß-glucuronidase (vestronidase alfa) was recently approved for use in patients; however, to date there have been no studies evaluating therapeutic efficacy in a large animal model of MPS VII. The objective of this study was to establish the effects of intravenous ERT, administered at either the standard clinical dose (4 mg/kg) or a high dose (20 mg/kg), on skeletal disease progression in MPS VII using the naturally occurring canine model. Untreated MPS VII animals exhibited progressive synovial joint and vertebral bone disease and were no longer ambulatory by age 6 months. Standard-dose ERT-treated animals exhibited modest attenuation of joint disease, but by age 6 months were no longer ambulatory. High-dose ERT-treated animals exhibited marked attenuation of joint disease, and all were still ambulatory by age 6 months. Vertebral bone disease was recalcitrant to ERT irrespective of dose. Overall, our findings indicate that ERT administered at higher doses results in significantly improved skeletal disease outcomes in MPS VII dogs.

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.

Experimental characterization and finite element implementation of soft tissue nonlinear viscoelasticity.

Finite element (FE) models of articular joint structures do not typically implement the fully nonlinear viscoelastic behavior of the soft connective tissue components. Instead, contemporary whole joint FE models usually represent the transient soft tissue behavior with significantly simplified formulations that are computationally tractable. The resultant fidelity of these models is greatly compromised with respect to predictions under temporally varying static and dynamic loading regimes. In addition, models based upon experimentally derived nonlinear viscoelastic coefficients that do not account for the transient behavior during the loading event(s) may further reduce the model's predictive accuracy. The current study provides the derivation and validation of a novel, phenomenological nonlinear viscoelastic formulation (based on the single integral nonlinear superposition formulation) that can be directly inputted into FE algorithms. This formulation and an accompanying experimental characterization technique, which incorporates relaxation manifested during the loading period of stress relaxation experiments, is compared to a previously published characterization method and validated against an independent analytical model. The results demonstrated that the static and dynamic FE approximations are in good agreement with the analytical solution. Additionally, the predictive accuracy of these approximations was observed to be highly dependent upon the experimental characterization technique. It is expected that implementation of the novel, computationally tractable nonlinear viscoelastic formulation and associated experimental characterization technique presented in the current study will greatly improve the predictive accuracy of the individual connective tissue components for whole joint FE simulations subjected to static and dynamic loading regimes.

Achilles Tendon Ruptures in Middle-Aged Rats Heal Poorly Compared With Those in Young and Old Rats [Formula: see text].

BACKGROUND: Achilles tendon ruptures are painful and debilitating injuries and are most common in middle-aged patients. There is a lack of understanding of the underlying causes for increased rupture rates in middle-aged patients and how healing outcomes after a rupture might be affected by patient age. Therefore, the objective of this study was to define age-specific Achilles tendon healing by assessing ankle functional outcomes and Achilles tendon mechanical and histological properties after a rupture using a rat model. HYPOTHESIS: Rats representing the middle-aged patient population would demonstrate reduced healing capability after an Achilles tendon rupture, as demonstrated by a slower return to baseline ankle functional properties and inferior biomechanical and histological tendon properties. STUDY DESIGN: Controlled laboratory study. METHODS: Fischer 344 rats were categorized by age to represent young, middle-aged, and old patients, and Achilles tendon ruptures were induced in the right hindlimb. Animals were allowed to heal and were euthanized at 3 or 6 weeks after the injury. In vivo functional assays and ultrasound imaging were performed throughout the healing period, and ex vivo tendon mechanical and histological properties were assessed after euthanasia. RESULTS: Rats representing middle-aged patients displayed reduced healing potential compared with the other age groups, as they demonstrated decreased recovery of in vivo functional and ultrasound assessment parameters and inferior mechanical and histological properties after an Achilles tendon rupture. CONCLUSION: These findings may help explain the increased rupture rate observed clinically in middle-aged patients by suggesting that there may be altered tendon responses to daily trauma. CLINICAL RELEVANCE: The results provide novel data on age-specific healing outcomes after an Achilles tendon rupture, which underscores the importance of considering a patient's age during treatment and expectations for outcomes.

Biglycan has a major role in maintenance of mature tendon mechanics.

Decorin and biglycan are two small leucine-rich proteoglycans (SLRPs) that regulate collagen fibrillogenesis and extracellular matrix assembly in tendon. The objective of this study was to determine the individual roles of these molecules in maintaining the structural and mechanical properties of tendon during homeostasis in mature mice. We hypothesized that knockdown of decorin in mature tendons would result in detrimental changes to tendon structure and mechanics while knockdown of biglycan would have a minor effect on these parameters. To achieve this objective, we created tamoxifen-inducible mouse knockdown models targeting decorin or biglycan inactivation. This enables the evaluation of the roles of these SLRPs in mature tendon without the abnormal tendon development caused by conventional knockout models. Contrary to our hypothesis, knockdown of decorin resulted in minor alterations to tendon structure and no changes to mechanics while knockdown of biglycan resulted in broad changes to tendon structure and mechanics. Specifically, knockdown of biglycan resulted in reduced insertion modulus, maximum stress, dynamic modulus, stress relaxation, and increased collagen fiber realignment during loading. Knockdown of decorin and biglycan produced similar changes to tendon microstructure by increasing the collagen fibril diameter relative to wild-type controls. Biglycan knockdown also decreased the cell nuclear aspect ratio, indicating a more spindle-like nuclear shape. Overall, the extensive changes to tendon structure and mechanics after knockdown of biglycan, but not decorin, provides evidence that biglycan plays a major role in the maintenance of tendon structure and mechanics in mature mice during homeostasis.

The Non-pregnant and Pregnant Human Cervix: a Systematic Proteomic Analysis.

Appropriate timing of cervical remodeling (CR) is key to normal term parturition. To date, mechanisms behind normal and abnormal (premature or delayed) CR remain unclear. Recent studies show regional differences exist in human cervical tissue structure. While the entire cervix contains extracellular matrix (ECM), the internal os is highly cellular containing 50-60% cervical smooth muscle (CSM). The external os contains 10-20% CSM. Previously, we reported ECM rigidity and different ECM proteins influence CSM cell function, highlighting the importance of understanding not only how cervical cells orchestrate cervical ECM remodeling in pregnancy, but also how changes in specific ECM proteins can influence resident cellular function. To understand this dynamic process, we utilized a systematic proteomic approach to understand which soluble ECM and cellular proteins exist in the different regions of the human cervix and how the proteomic profiles change from the non-pregnant (NP) to the pregnant (PG) state. We found the human cervix proteome contains at least 4548 proteins and establish the types and relative abundance of cellular and soluble matrisome proteins found in the NP and PG human cervix. Further, we report the relative abundance of proteins involved with elastic fiber formation and ECM organization/degradation were significantly increased while proteins involved in RNA polymerase I/promoter opening, DNA methylation, senescence, immune system, and compliment activation were decreased in the PG compared to NP cervix. These findings establish an initial platform from which we can further comprehend how changes in the human cervix proteome results in normal and abnormal CR.

Inhibition of glucose use improves structural recovery of injured Achilles tendon in mice.

Injured tendons do not regain their native structure except at fetal or very young ages. Healing tendons often show mucoid degeneration involving accumulation of sulfated glycosaminoglycans (GAGs), but its etiology and molecular base have not been studied substantially. We hypothesized that quality and quantity of gene expression involving the synthesis of proteoglycans having sulfated GAGs are altered in injured tendons and that a reduction in synthesis of sulfated GAGs improves structural and functional recovery of injured tendons. C57BL6/j mice were subjected to Achilles tendon tenotomy surgery. The injured tendons accumulated sulfate proteoglycans as early as 1-week postsurgery and continued so by 4-week postsurgery. Transcriptome analysis revealed upregulation of a wide range of proteoglycan genes that have sulfated GAGs in the injured tendons 1 and 3 weeks postsurgery. Genes critical for enzymatic reaction of initiation and elongation of chondroitin sulfate GAG chains were also upregulated. After the surgery, mice were treated with the 2-deoxy-d-glucose (2DG) that inhibits conversion of glucose to glucose-6-phosphate, an initial step of glucose metabolism as an energy source and precursors of monosaccharides of GAGs. The 2DG treatment reduced accumulation of sulfated proteoglycans, improved collagen fiber alignment, and reduced the cross-sectional area of the injured tendons. The modulus of the 2DG-treated groups was higher than that in the vehicle group, but not of statistical significance. Our findings suggest that mucoid degeneration in injured tendons may result from the upregulated expression of genes involved the synthesis of sulfate proteoglycans and can be inhibited by reduction of glucose utilization.

Coordinate roles for collagen VI and biglycan in regulating tendon collagen fibril structure and function.

Tendon is a vital musculoskeletal tissue that is prone to degeneration. Proper tendon maintenance requires complex interactions between extracellular matrix components that remain poorly understood. Collagen VI and biglycan are two matrix molecules that localize pericellularly within tendon and are critical regulators of tissue properties. While evidence suggests that collagen VI and biglycan interact within the tendon matrix, the relationship between the two molecules and its impact on tendon function remains unknown. We sought to elucidate potential coordinate roles of collagen VI and biglycan within tendon by defining tendon properties in knockout models of collagen VI, biglycan, or both molecules. We first demonstrated co-expression and co-localization of collagen VI and biglycan within the healing tendon, providing further evidence of cooperation between the two molecules during nascent tendon matrix formation. Deficiency in collagen VI and/or biglycan led to significant reductions in collagen fibril size and tendon mechanical properties. However, collagen VI-null tendons displayed larger reductions in fibril size and mechanics than seen in biglycan-null tendons. Interestingly, knockout of both molecules resulted in similar properties to collagen VI knockout alone. These results indicate distinct and non-additive roles for collagen VI and biglycan within tendon. This work provides better understanding of regulatory interactions between two critical tendon matrix molecules.

The Inner Annulus Fibrosus Encroaches on the Nucleus Pulposus in the Injured Mouse Tail Intervertebral Disc.

OBJECTIVE: The aim was to identify the source of cells within the center of the abnormal fibrocartilage tissue of the degenerative intervertebral disc after injury. DESIGN: Cross-breeding of mice with an inducible type II promoter collagen construct (Col2CreER) to Rosa26-TdTomato mice has been shown to result in Cre-recombinase activity and Tomato expression in inner annulus fibrosus cells after tamoxifen injection. To investigate the role of the inner annulus fibrosus in the intervertebral disc injury response, tail intervertebral discs of Col2CreER/tdTomato mice were punctured with a needle and examined 1-4 wks after injury. N-cadherin was examined by immunostaining. RESULTS: After the injury, the fibrocartilage in the degenerative intervertebral disc consisted of residual diseased nucleus pulposus cells and encroaching inner annulus fibrosus cells. The residual nucleus pulposus cells had lost their epithelial cell-like morphology and instead became oval shaped, with reduced adhesion to neighboring nucleus pulposus cells. This change in cellular morphology coincided with a loss of N-cadherin, which contributes to maintenance of healthy nucleus pulposus cell morphology. As expected, injured tail intervertebral discs showed reduced compressive properties as determined by biomechanical assessments. CONCLUSIONS: The cellular composition of the degenerative intervertebral disc has been defined here, which is an important step in developing future treatments.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation.

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.

Evaluation of Autologous Protein Solution Injection for Treatment of Superficial Digital Flexor Tendonitis in an Equine Model.

Autologous protein solution (APS) has been used anecdotally for intralesional treatment of tendon and ligament injuries, however, its use in these injuries has never been studied in vivo. Our objective was to evaluate the effect of APS on tendon healing in an equine superficial digital flexor (SDF) tendonitis model. We hypothesized intralesional injection of APS would result in superior structural and biomechanical healing. SDF tendonitis was induced in both forelimbs of eight horses using collagenase injection. One forelimb was randomly assigned to receive an intralesional injection of APS, while the other was injected with saline. Ultrasonographic examinations were performed at weeks -1, 0, 2, 4, 8, and 12 following treatment. At 12 weeks, horses were euthanized and SDF samples harvested. Histologic evaluation, biomechanical testing, gene expression analysis, total glycosaminoglycan (GAG) and total DNA quantification were performed. Collagen type III (COL3A1) expression was significantly higher (p = 0.028) in saline treated tendon than in normal tendon. Otherwise, there were no significant differences in gene expression. There were no significant differences in histologic or ultrasonographic scores between groups. Mean total DNA content was significantly higher (p = 0.024) in saline treated tendons than normal tendons, whereas total DNA content was not significantly different between APS treated tendon and normal tendon. Elastic modulus was higher in APS treated than saline treated tendon, but the difference was not significant. Reduced expression of COL3A1 in APS treated tendon may indicate superior healing. Increased total DNA content in saline treated tendon may indicate ongoing healing processes, vs. APS treated tendons which may be in the later stages of healing. Limitations include a relatively short study period and inconsistency in size and severity of induced lesions. Intralesional injection of APS resulted in some improvements in healing characteristics.

MRI-derived porosity index is associated with whole-bone stiffness and mineral density in human cadaveric femora.

Ultrashort echo time (UTE) magnetic resonance imaging (MRI) measures proton signals in cortical bone from two distinct water pools, bound water, or water that is tightly bound to bone matrix, and pore water, or water that is freely moving in the pore spaces in bone. By isolating the signal contribution from the pore water pool, UTE biomarkers can directly quantify cortical bone porosity in vivo. The Porosity Index (PI) is one non-invasive, clinically viable UTE-derived technique that has shown strong associations in the tibia with µCT porosity and other UTE measures of bone water. However, the efficacy of the PI biomarker has never been examined in the proximal femur, which is the site of the most catastrophic osteoporotic fractures. Additionally, the loads experienced during a sideways fall are complex and the femoral neck is difficult to image with UTE, so the usefulness of the PI in the femur was unknown. Therefore, the aim of this study was to examine the relationships between the PI measure in the proximal cortical shaft of human cadaveric femora specimens compared to (1) QCT-derived bone mineral density (BMD) and (2) whole bone stiffness obtained from mechanical testing mimicking a sideways fall. Fifteen fresh, frozen whole cadaveric femora specimens (age 72.1 ± 15.0 years old, 10 male, 5 female) were scanned on a clinical 3-T MRI using a dual-echo UTE sequence. Specimens were then scanned on a clinical CT scanner to measure volumetric BMD (vBMD) and then non-destructively mechanically tested in a sideways fall configuration. The PI in the cortical shaft demonstrated strong correlations with bone stiffness (r = -0.82, P = 0.0014), CT-derived vBMD (r = -0.64, P = 0.0149), and with average cortical thickness (r = -0.60, P = 0.0180). Furthermore, a hierarchical regression showed that PI was a strong predictor of bone stiffness which was independent of the other parameters. The findings from this study validate the MRI-derived porosity index as a useful measure of whole-bone mechanical integrity and stiffness.

Collagen XI regulates the acquisition of collagen fibril structure, organization and functional properties in tendon.

Collagen XI is a fibril-forming collagen that regulates collagen fibrillogenesis. Collagen XI is normally associated with collagen II-containing tissues such as cartilage, but it also is expressed broadly during development in collagen I-containing tissues, including tendons. The goals of this study are to define the roles of collagen XI in regulation of tendon fibrillar structure and the relationship to function. A conditional Col11a1-null mouse model was created to permit the spatial and temporal manipulation of Col11a1 expression. We hypothesize that collagen XI functions to regulate fibril assembly, organization and, therefore, tendon function. Previous work using cho mice with ablated Col11a1 alleles supported roles for collagen XI in tendon fibril assembly. Homozygous cho/cho mice have a perinatal lethal phenotype that limited the studies. To circumvent this, a conditional Col11a1flox/flox mouse model was created where exon 3 was flanked with loxP sites. Breeding with Scleraxis-Cre (Scx-Cre) mice yielded a tendon-specific Col11a1-null mouse line, Col11a1Δten/Δten. Col11a1flox/flox mice had no phenotype compared to wild type C57BL/6 mice and other control mice, e.g., Col11a1flox/flox and Scx-Cre. Col11a1flox/flox mice expressed Col11a1 mRNA at levels comparable to wild type and Scx-Cre mice. In contrast, in Col11a1Δten/Δten mice, Col11a1 mRNA expression decreased to baseline in flexor digitorum longus tendons (FDL). Collagen XI protein expression was absent in Col11a1Δten/Δten FDLs, and at ~50% in Col11a1+/Δten compared to controls. Phenotypically, Col11a1Δten/Δten mice had significantly decreased body weights (p < 0.001), grip strengths (p < 0.001), and with age developed gait impairment becoming hypomobile. In the absence of Col11a1, the tendon collagen fibrillar matrix was abnormal when analyzed using transmission electron microscopy. Reducing Col11a1 and, therefore collagen XI content, resulted in abnormal fibril structure, loss of normal fibril diameter control with a significant shift to small diameters and disrupted parallel alignment of fibrils. These alterations in matrix structure were observed in developing (day 4), maturing (day 30) and mature (day 60) mice. Altering the time of knockdown using inducible I-Col11a1-/- mice indicated that the primary regulatory foci for collagen XI was in development. In mature Col11a1Δten/Δten FDLs a significant decrease in the biomechanical properties was observed. The decrease in maximum stress and modulus suggest that fundamental differences in the material properties in the absence of Col11a1 expression underlie the mechanical deficiencies. These data demonstrate an essential role for collagen XI in regulation of tendon fibril assembly and organization occurring primarily during development.

Amplifying Bone Marrow Progenitors Expressing α-Smooth Muscle Actin Produce Zonal Insertion Sites During Tendon-to-Bone Repair.

Traditional tendon-to-bone repair where the tendon is reattached to bone via suture anchors often results in disorganized scar production rather than the formation of a zonal insertion. In contrast, ligament reconstructions where tendon grafts are passed through bone tunnels can yield zonal tendon-to-bone attachments between the graft and adjacent bone. Therefore, ligament reconstructions can be used to study mechanisms that regulate zonal tendon-to-bone repair in the adult. Anterior cruciate ligament (ACL) reconstructions are one of the most common reconstruction procedures and while we know that cells from outside the graft produce the attachments, we have not yet established specific cell populations that give rise to this tissue. To address this knowledge gap, we performed ACL reconstructions in lineage tracing mice where α-smooth muscle actin (αSMACreERT2) was used to label αSMA-expressing progenitors within the bone marrow that produced zonal attachments. Expression of αSMA was increased during early stages of the repair process such that the contribution of SMA-labeled cells to the tunnel integration was highest when tamoxifen was delivered in the first week post-surgery. The zonal attachments shared features with normal entheses, including tidemarks oriented perpendicularly to collagen fibers, Col1a1-expressing cells, alkaline phosphatase activity, and proteoglycan-rich staining. Finally, the integration strength increased with time, requiring 112% greater force to remove the graft from the tunnel at 28 days compared with 14 days post-surgery. Future studies will target these progenitor cells to define the pathways that regulate zonal tendon-to-bone repair in the adult. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:105-116, 2020.