A role for TGFß signaling in Gli1+ tendon and enthesis cells.

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFß signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFß signaling in Gli1+ cell function, the receptor for TGFß, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFß signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFß signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFß controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFß directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

Engineered Microenvironmental Cues from Fiber-Reinforced Hydrogel Composites Drive Tenogenesis and Aligned Collagen Deposition.

Effective tendon regeneration following injury is contingent on appropriate differentiation of recruited cells and deposition of mature, aligned, collagenous extracellular matrix that can withstand the extreme mechanical demands placed on the tissue. As such, myriad biomaterial approaches have been explored to provide biochemical and physical cues that encourage tenogenesis and template aligned matrix deposition in lieu of dysfunctional scar tissue formation. Fiber-reinforced hydrogels present an ideal biomaterial system toward this end given their transdermal injectability, tunable stiffness over a range amenable to tenogenic differentiation of progenitors, and capacity for modular inclusion of biochemical cues. Here, tunable and modular, fiber-reinforced, synthetic hydrogels are employed to elucidate salient microenvironmental determinants of tenogenesis and aligned collagen deposition by tendon progenitor cells. Transforming growth factor ß3 drives a cell fate switch toward pro-regenerative or pro-fibrotic phenotypes, which can be biased toward the former by culture in softer microenvironments or inhibition of the RhoA/ROCK activity. Furthermore, studies demonstrate that topographical anisotropy in fiber-reinforced hydrogels critically mediates the alignment of de novo collagen fibrils, reflecting native tendon architecture. These findings inform the design of cell-free, injectable, synthetic hydrogels for tendon tissue regeneration and, likely, that of a range of load-bearing connective tissues.

Interfacial Tissue Regeneration with Bone.

PURPOSE OF REVIEW: Interfacial tissue exists throughout the body at cartilage-to-bone (osteochondral interface) and tendon-to-bone (enthesis) interfaces. Healing of interfacial tissues is a current challenge in regenerative approaches because the interface plays a critical role in stabilizing and distributing the mechanical stress between soft tissues (e.g., cartilage and tendon) and bone. The purpose of this review is to identify new directions in the field of interfacial tissue development and physiology that can guide future regenerative strategies for improving post-injury healing. RECENT FINDINGS: Cues from interfacial tissue development may guide regeneration including biological cues such as cell phenotype and growth factor signaling; structural cues such as extracellular matrix (ECM) deposition, ECM, and cell alignment; and mechanical cues such as compression, tension, shear, and the stiffness of the cellular microenvironment. In this review, we explore new discoveries in the field of interfacial biology related to ECM remodeling, cellular metabolism, and fate. Based on emergent findings across multiple disciplines, we lay out a framework for future innovations in the design of engineered strategies for interface regeneration. Many of the key mechanisms essential for interfacial tissue development and adaptation have high potential for improving outcomes in the clinic.

Nuclear Factor Kappa B Over-Activation in the Intervertebral Disc Leads to Macrophage Recruitment and Severe Disc Degeneration.

Objective: Low back pain (LBP) is the leading cause of global disability and is thought to be driven primarily by intervertebral disc (IVD) degeneration (DD). Persistent upregulation of catabolic enzymes and inflammatory mediators have been associated with severe cases of DD. Nuclear factor kappa B (NF-κB) is a master transcription regulator of immune responses and is over expressed during inflammatory-driven musculoskeletal diseases, including DD. However, its role in triggering DD is unknown. Therefore, this study investigated the effect of NF-κB pathway over-activation on IVD integrity and DD pathology. Methods: Using skeletally mature mouse model, we genetically targeted IVD cells for canonical NF-κB pathway activation via expression of a constitutively active form of inhibitor of κB kinase B (IKKß), and assessed changes in IVD cellularity, structural integrity including histology, disc height, and extracellular matrix (ECM) biochemistry, biomechanics, expression of inflammatory, catabolic, and neurotropic mediators, and changes in macrophage subsets, longitudinally up to 6-months post activation. Results: Prolonged NF-κB activation led to severe structural degeneration, with a loss of glycosaminoglycan (GAG) content and complete loss of nucleus pulposus (NP) cellularity. Structural and compositional changes decreased IVD height and compressive mechanical properties with prolonged NF-κB activation. These alterations were accompanied by increases in gene expression of inflammatory molecules ( Il1b, Il6, Nos2 ), chemokines ( Mcp1 , Mif ), catabolic enzymes ( Mmp3, Mmp9, Adamts4 ), and neurotrophic factors ( Bdnf , Ngf ) within IVD tissue. Increased recruitment of activated F4/80 + macrophages exhibited a greater abundance of pro-inflammatory (CD38 + ) over inflammatory-resolving (CD206 + ) macrophage subsets in the IVD, with temporal changes in the relative abundance of macrophage subsets over time, providing evidence for temporal regulation of macrophage polarization in DD in vivo, where macrophages participate in resolving the inflammatory cascade but promote fibrotic transformation of the IVD matrix. We further show that NF-κB driven secretory factors from IVD cells increase macrophage migration and inflammatory activation, and that the secretome of inflammatory-resolving macrophages mitigates effects of NF-κB overactivation. Conclusion: Overall the observed results suggest prolonged NF-κB activation can induce severe DD, acting through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and inflammatory activation of a macrophage cell populations, that can be mitigated with inflammatory-resolving macrophage secretome.

Optogenetic-induced muscle loading leads to mechanical adaptation of the Achilles tendon enthesis in mice.

Skeletal shape depends on the transmission of contractile muscle forces from tendon to bone across the enthesis. Loss of muscle loading impairs enthesis development, yet little is known if and how the postnatal enthesis adapts to increased loading. Here, we studied adaptations in enthesis structure and function in response to increased loading, using optogenetically induced muscle contraction in young (i.e., growth) and adult (i.e., mature) mice. Daily bouts of unilateral optogenetic loading in young mice led to radial calcaneal expansion and warping. This also led to a weaker enthesis with increased collagen damage in young tendon and enthisis, with little change in adult mice. We then used RNA sequencing to identify the pathways associated with increased mechanical loading during growth. In tendon, we found enrichment of glycolysis, focal adhesion, and cell-matrix interactions. In bone, we found enrichment of inflammation and cell cycle. Together, we demonstrate the utility of optogenetic-induced muscle contraction to elicit in vivo adaptation of the enthesis.

Optogenetic-Induced Muscle Loading Leads to Mechanical Adaptation of the Achilles Tendon Enthesis in Mice.

The growth of the skeleton depends on the transmission of contractile muscle forces from tendon to bone across the extracellular matrix-rich enthesis. Loss of muscle loading leads to significant impairments in enthesis development. However, little is known about how the enthesis responds to increased loading during postnatal growth. To study the cellular and matrix adaptations of the enthesis in response to increased muscle loading, we used optogenetics to induce skeletal muscle contraction and unilaterally load the Achilles tendon and enthesis in young (i.e., during growth) and adult (i.e., mature) mice. In young mice, daily bouts of unilateral optogenetic loading led to expansion of the calcaneal apophysis and growth plate, as well as increased vascularization of the normally avascular enthesis. Daily loading bouts, delivered for 3 weeks, also led to a mechanically weaker enthesis with increased molecular-level accumulation of collagen damage in young mice. However, adult mice did not exhibit impaired mechanical properties or noticeable structural adaptations to the enthesis. We then focused on the transcriptional response of the young tendon and bone following optogenetic-induced loading. After 1 or 2 weeks of loading, we identified, in tendon, transcriptional activation of canonical pathways related to glucose metabolism (glycolysis) and inhibited pathways associated with cytoskeletal remodeling (e.g., RHOA and CREB signaling). In bone, we identified activation of inflammatory signaling (e.g., NFkB and STAT3 signaling) and inhibition of ERK/MAPK and PTEN signaling. Thus, we have demonstrated the utility of optogenetic-induced skeletal muscle contraction to elicit structural, functional, and molecular adaptation of the enthesis in vivo especially during growth.

Comparison of Distal Spine Anchors and Distal Pelvic Anchors in Children With Hypotonic Neuromuscular Scoliosis Treated With Growth-friendly Instrumentation.

BACKGROUND: Lower preoperative pelvic obliquity (PO) and L5 tilt have been associated with good radiographic outcomes when the fusion ended short of the pelvis in children with neuromuscular scoliosis (NMS). Our purpose was to identify indications to exclude the pelvis in children with hypotonic NMS treated with growth-friendly instrumentation. METHODS: This was a multicenter retrospective review. Children with spinal muscular atrophy and muscular dystrophy treated with dual traditional growing rod, magnetically controlled growing rod, or vertical expandable prosthetic titanium rib with minimum 2-year follow-up after the index surgery were identified. RESULTS: A total of 125 patients met the inclusion criteria. Thirty-eight patients had distal spine anchors (DSAs) and 87 patients had distal pelvic anchors (DPAs) placed at the index surgery. Demographics and length of follow-up were similar between the groups but there was a greater percentage of DPA patients who were nonambulatory [79 patients (91%) vs. 18 patients (47%), P <0.0001]. Preindex radiographic measures were similar except the DSA patients had a lower PO (11 vs. 19 degrees, P =0.0001) and L5 tilt (8 vs. 12 degrees, P =0.001). Postindex and most recent radiographic data were comparable between the groups. There was no difference in the complication and unplanned returns to the operating room rates.Subanalysis of the DSA group based on ambulatory status showed similar radiographic measures except the ambulatory patients had a lower PO at all time points (preindex: 5 vs. 16 degrees, P =0.011; postindex: 6 vs. 10 degrees, P =0.045; most recent follow-up: 5 vs. 14 degrees, P =0.028). Only 1 ambulatory DSA patient had a PO ≥10 degrees at most recent follow-up compared with 6 nonambulatory DSA patients. Three (8%) DSA patients, all nonambulatory, underwent extension of their instrumentation to the pelvis. CONCLUSIONS: Pelvic fixation should be strongly considered in nonambulatory children with hypotonic NMS treated with growth-friendly instrumentation. At intermediate-term follow-up, revision surgery to include the pelvis was rare but DSAs do not seem effective at maintaining control of PO in nonambulatory patients. DSA and DPA were equally effective at maintaining major curve control, and complication and unplanned returns to the operating room rates were similar. LEVEL OF EVIDENCE: Level III-therapeutic.

Tendon progenitor cells as biological augmentation improve functional gait and reduce scar formation after rotator cuff repair.

BACKGROUND: High rates of structural failure are reported after rotator cuff repairs due to inability to recreate the native enthesis during healing. The development of biological augmentation methods that mitigate scar formation and regenerate the enthesis is still an unmet need. Since neonatal enthesis is capable of regeneration after injury, this study tested whether delivery of neonatal tendon progenitor cells (TPCs) into the adult injured environment can enhance functional and structural supraspinatus enthesis and tendon healing. METHODS: TPCs were isolated from Ai14 Rosa26-TdTomato mouse Achilles tendons and labeled using adenovirus-Cre. Fifty-two CB57BL/6J mice underwent detachment and acute repair of the supraspinatus tendon and received either a fibrin-only or TPC-fibrin gel. Immunofluorescence analysis was carried out to determine cellularity (DAPI), fibrocartilage (SOX9), macrophages (F4/80), myofibroblasts (α-smooth muscle actin), and scar (laminin). Assays for function (gait and biomechanical testing) and structure (micro-computed tomography imaging, picrosirius red/Alcian Blue staining, type I and III collagen staining) were carried out. RESULTS: Analysis of TdTomato cells after injury showed minimal retention of TPCs by day 7 and day 14, with detected cells localized near the bursa and deltoid rather than the enthesis/tendon. However, TPC delivery led to significantly increased %Sox9+ cells in the enthesis at day 7 after injury and decreased laminin intensity across almost all time points compared to fibrin-only treatment. Similarly, TPC-treated mice showed gait recovery by day 14 (paw area and stride length) and day 28 (stance time), while fibrin-treated mice failed to recover gait parameters. Despite improved gait, biomechanical testing showed no differences between groups. Structural analysis by micro-computed tomography suggests that TPC application improves cortical thickness after surgery compared to fibrin. Superior collagen alignment at the neo-enthesis was also observed in the TPC-augmented group at day 28, but no difference was detected in type I and III collagen intensity. CONCLUSION: We found that neonatal TPCs improved and restored functional gait by reducing overall scar formation, improving enthesis collagen alignment, and altering bony composition response after supraspinatus tendon repair. TPCs did not appear to integrate into the healing tissue, suggesting improved healing may be due to paracrine effects at early stages. Future work will determine the factors secreted by TPCs to develop translational targets.

The role of loading in murine models of rotator cuff disease.

Rotator cuff disease pathogenesis is associated with intrinsic (e.g., age, joint laxity, muscle weakness) and extrinsic (e.g., mechanical load, fatigue) factors that lead to chronic degeneration of the cuff tissues. However, etiological studies are difficult to perform in patients due to the long duration of disease onset and progression. Therefore, the purpose of this study was to determine the effects of altered joint loading on the rotator cuff. Mice were subjected to one of three load-dependent rotator cuff tendinopathy models: underuse loading, achieved by injecting botulinum toxin-A into the supraspinatus muscle; overuse loading, achieved using downhill treadmill running; destabilization loading, achieved by surgical excision of the infraspinatus tendon. All models were compared to cage activity animals. Whole joint function was assessed longitudinally using gait analysis. Tissue-scale structure and function were determined using microCT, tensile testing, and histology. The molecular response of the supraspinatus tendon and enthesis was determined by measuring the expression of 84 wound healing-associated genes. Underuse and destabilization altered forepaw weight-bearing, decreased tendon-to-bone attachment strength, decreased mineral density of the humeral epiphysis, and reduced tendon strength. Transcriptional activity of the underuse group returned to baseline levels by 4 weeks, while destabilization had significant upregulation of inflammation, growth factors, and extracellular matrix remodeling genes. Surprisingly, overuse activity caused changes in walking patterns, increased tendon stiffness, and primarily suppressed expression of wound healing-related genes. In summary, the tendinopathy models demonstrated how divergent muscle loading can result in clinically relevant alterations in rotator cuff structure, function, and gene expression.

Toughening mechanisms for the attachment of architectured materials: The mechanics of the tendon enthesis.

Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and previously unexplored imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering.

Enhanced Tendon-to-Bone Healing via IKKß Inhibition in a Rat Rotator Cuff Model.

BACKGROUND: More than 450,000 rotator cuff repairs are performed annually, yet healing of tendon to bone often fails. This failure is rooted in the fibrovascular healing response, which does not regenerate the native attachment site. Better healing outcomes may be achieved by targeting inflammation during the early period after repair. Rather than broad inhibition of inflammation, which may impair healing, the current study utilized a molecularly targeted approach to suppress IKKß, shutting down only the inflammatory arm of the nuclear factor κB (NF-κB) signaling pathway. PURPOSE: To evaluate the therapeutic potential of IKKß inhibition in a clinically relevant model of rat rotator cuff repair. STUDY DESIGN: Controlled laboratory study. METHODS: After validating the efficacy of the IKKß inhibitor in vitro, it was administered orally once a day for 7 days after surgery in a rat rotator cuff repair model. The effect of treatment on reducing inflammation and improving repair quality was evaluated after 3 days and 2, 4, and 8 weeks of healing, using gene expression, biomechanics, bone morphometry, and histology. RESULTS: Inhibition of IKKß attenuated cytokine and chemokine production in vitro, demonstrating the potential for this inhibitor to reduce inflammation in vivo. Oral treatment with IKKß inhibitor reduced NF-κB target gene expression by up to 80% compared with a nontreated group at day 3, with a subset of these genes suppressed through 14 days. Furthermore, the IKKß inhibitor led to enhanced tenogenesis and extracellular matrix production, as demonstrated by gene expression and histological analyses. At 4 weeks, inhibitor treatment led to increased toughness, no effects on failure load and strength, and decreases in stiffness and modulus when compared with vehicle control. At 8 weeks, IKKß inhibitor treatment led to increased toughness, failure load, and strength compared with control animals. IKKß inhibitor treatment prevented the bone loss near the tendon attachment that occurred in repairs in control. CONCLUSION: Pharmacological inhibition of IKKß successfully suppressed excessive inflammation and enhanced tendon-to-bone healing after rotator cuff repair in a rat model. CLINICAL RELEVANCE: The NF-κB pathway is a promising target for enhancing outcomes after rotator cuff repair.

Cell lineage tracing and functional assessment of supraspinatus tendon healing in an acute repair murine model.

Rotator cuff supraspinatus tendon injuries are common with high rates of anatomic failure after surgical repair. The purpose of the study was to define clinically relevant features of a mouse model of supraspinatus tendon injury to determine painful, functional, and structural outcomes; we further investigated two cell populations mediating healing using genetic lineage tracing after full detachment and repair of the supraspinatus tendon in mice. The pain was assessed using the mouse grimace scale and function by gait analysis and tensile testing. Histological and microCT analyses were used to determine enthesis/tendon and bone structure, respectively. Lineage tracing was carried out using inducible Cre lines for ScxCreERT2 (tendon cells) and αSMACreERT2 (myofibroblasts and mesenchymal progenitors). Mice only expressed pain transiently after surgery despite long-term impairment of functional and structural properties. Gait, tensile mechanical properties, and bone properties were significantly reduced after injury and repair. Lineage tracing showed relatively few Scx lin tendon cells while αSMA lin cells contributed strongly to scar formation. Despite surgical reattachment of healthy tendon, lineage tracing revealed poor preservation of supraspinatus tendon after acute injury and loss of tendon structure, suggesting that tendon degeneration is also a key impediment of successful rotator cuff repair. Scar formation after surgery is mediated largely by αSMA lin cells and results in permanently reduced functional and structural properties.

Biomechanical Testing of Murine Tendons.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

Targeting the NF-κB signaling pathway in chronic tendon disease.

Tendon disorders represent the most common musculoskeletal complaint for which patients seek medical attention; inflammation drives tendon degeneration before tearing and impairs healing after repair. Clinical evidence has implicated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway as a correlate of pain-free return to function after surgical repair. However, it is currently unknown whether this response is a reaction to or a driver of pathology. Therefore, we aimed to understand the clinically relevant involvement of the NF-κB pathway in tendinopathy, to determine its potential causative roles in tendon degeneration, and to test its potential as a therapeutic candidate. Transcriptional profiling of early rotator cuff tendinopathy identified increases in NF-κB signaling, including increased expression of the regulatory serine kinase subunit IKKß, which plays an essential role in inflammation. Using cre-mediated overexpression of IKKß in tendon fibroblasts, we observed degeneration of mouse rotator cuff tendons and the adjacent humeral head. These changes were associated with increases in proinflammatory cytokines and innate immune cells within the joint. Conversely, genetic deletion of IKKß in tendon fibroblasts partially protected mice from chronic overuse-induced tendinopathy. Furthermore, conditional knockout of IKKß improved outcomes after surgical repair, whereas overexpression impaired tendon healing. Accordingly, targeting of the IKKß/NF-κB pathway in tendon stromal cells may offer previously unidentified therapeutic approaches in the management of human tendon disorders.

Local bone quality measurements correlates with maximum screw torque at the femoral diaphysis.

BACKGROUND: Successful fracture fixation depends critically on the stability of the screw-bone interface. Maximum achievable screw torque reflects the competence of this interface, but it cannot be quantified prior to screw stripping. Typically, the surgeon relies on the patients' bone mineral density and radiographs, along with experience and tactile feedback to assess whether sufficient compression can be generated by the screw and bone. However, the local bone quality would also critically influence the strength of the bone-screw interface. We investigated whether Reference Point Indentation can provide quantitative local bone quality measures that can inform subsequent screw-bone competence. METHODS: We examined the associations between the maximum screw torque that can be achieved using 3.5 mm, 4.5 mm, and 6.5 mm diameter stainless steel screws at the distal femoral metaphysis and mid-diaphysis from 20 cadavers, with the femoral neck bone mineral density and the local measures of bone quality using Reference Point Indentation. FINDINGS: Indentation Distance Increase, a measure of bone's resistance to microfracture, correlated with the maximum screw stripping torque for the 3.5 mm (p < 0.01; R = 0.56) and 4.5 mm diameter stainless steel screws (p < 0.01; R = 0.57) at the femoral diaphysis. At the femoral metaphysis, femoral neck bone mineral density significantly correlated with the maximum screw stripping torque achieved by the 3.5 mm (p < 0.01; R = 0.61), 4.5 mm (p < 0.01; R = 0.51), and 6.5 mm diameter stainless steel screws (p < 0.01; R = 0.56). INTERPRETATION: Reference Point Indentation can provide localized measurements of bone quality that may better inform surgeons of the competence of the bone-implant interface and improve effectiveness of fixation strategies particularly in patients with compromised bone quality.

Targeting Inflammation in Rotator Cuff Tendon Degeneration and Repair.

Rotator cuff degeneration is a common affliction that results in pain and disability. Tendinopathy was historically classified with or without the involvement of the immune system. However, technological advancements in screening have shown that the immune system is both present and active in all forms of tendinopathy. During injury and healing, the coordinated effort of numerous immune cell populations work with the resident stromal cells to break down damaged tissues and stimulate remodeling. These cells deploy a wide array of tools, including phagocytosis, enzyme secretion, and chemotactic gradients to direct these processes. Yet, there remains a knowledge gap in our understanding of the sequence of critical events and regulatory factors that mediate this is process in injury and healing. Furthermore, current treatments do not specifically target inflammation at the molecular level. Typical regimens include non-steroidal anti-inflammatory drugs or corticosteroids; however, researchers have found irrevocable functional deficits following treatment, and have disputed their long-term efficacy. Therefore, developing therapeutics that specifically consider the nuances of the immune system are necessary to improve patient outcomes.

Orthopedic Interface Repair Strategies Based on Native Structural and Mechanical Features of the Multiscale Enthesis.

The enthesis is an organ that connects a soft, aligned tissue (tendon/ligament) to a hard, amorphous tissue (bone) via a fibrocartilage interface. Mechanically, the enthesis sustains a dynamic loading environment that includes tensile, compressive, and shear forces. The structural components of the enthesis act to minimize stress concentrations and control stretch at the interface. Current surgical repair of the enthesis, such as in rotator cuff repair and anterior cruciate ligament reconstruction, aim to bridge the gap between the injured ends via reattachment of soft-to-hard tissues or graft replacement. In this review, we discuss the multiscale, morphological, and mechanical characteristics of the fibrocartilage attachment. Additionally, we review historical and recent clinical approaches to treating enthesis injury. Lastly, we explore new technological advancements in tissue-engineered biomaterials that have shown promise in preclinical studies.

Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing.

Scleraxis (Scx) is a known regulator of tendon development, and recent work has identified the role of Scx in bone modeling. However, the role of Scx in fracture healing has not yet been explored. This study was conducted to identify the role of Scx in cortical bone development and fracture healing. Scx green fluorescent protein-labeled (ScxGFP) reporter and Scx-knockout (Scx-mutant) mice were used to assess bone morphometry and the effects of fracture healing on Scx localization and gene expression, as well as callus healing response. Botulinum toxin (BTX) was used to investigate muscle unloading effects on callus shape. Scx-mutant long bones had structural and mechanical defects. Scx gene expression was elevated and bmp4 was decreased at 24 h after fracture. ScxGFP+ cells were localized throughout the healing callus after fracture. Scx-mutant mice demonstrated disrupted callus healing and asymmetry. Asymmetry of Scx-mutant callus was not due to muscle unloading. Wild-type littermates (age matched) served as controls. This is the first study to explore the role of Scx in cortical bone mechanics and fracture healing. Deletion of Scx during development led to altered long bone properties and callus healing. This study also demonstrated that Scx may play a role in the periosteal response during fracture healing.-McKenzie, J. A., Buettmann, E., Abraham, A. C., Gardner, M. J., Silva, M. J., Killian, M. L. Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing.

Longitudinal changes in the structure and inflammatory response of the intervertebral disc due to stab injury in a murine organ culture model.

Despite the significant public health impact of intervertebral disc (IVD) degeneration and low back pain, it remains challenging to investigate the multifactorial molecular mechanisms that drive the degenerative cascade. Organ culture model systems offer the advantage of allowing cells to live and interact with their native extracellular matrix, while simultaneously reducing the amount of biological variation and complexity present at the organismal level. Murine organ cultures in particular also allow the use of widely available genetically modified animals with molecular level reporters that would reveal insights on the degenerative cascade. Here, we utilize an organ culture system of murine lumbar functional spinal units where we are able to maintain the cellular, metabolic, and structural, and mechanical stability of the whole organ over a 21-day period. Furthermore, we describe a novel approach in organ culture by using tissues from animals with an NF-κB-luc reporter in combination with a mechanical injury model, and are able to show that proinflammatory factors and cytokines such as NF-κB and IL-6 produced by IVD cells can be monitored longitudinally during culture in a stab injury model. Taken together, we utilize a murine organ culture system that maintains the cellular and tissue level behavior of the intervertebral disc and apply it to transgenic animals that allow the monitoring of the inflammatory profile of IVDs. This approach could provide important insights on the molecular and metabolic mediators that regulate the homeostasis of the IVD. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1431-1438, 2016.

A 5 nW Quasi-Linear CMOS Hot-Electron Injector for Self-Powered Monitoring of Biomechanical Strain Variations.

Piezoelectricity-driven hot-electron injectors (p-HEI) are used for self-powered monitoring of mechanical activity in biomechanical implants and structures. Previously reported p-HEI devices operate by harvesting energy from a piezoelectric transducer to generate current and voltage references which are then used for initiating and controlling the process of hot-electron injection. As a result, the minimum energy required to activate the device is limited by the power requirements of the reference circuits. In this paper we present a p-HEI device that operates by directly exploiting the self-limiting capability of an energy transducer when driving the process of hot-electron injection in a pMOS floating-gate transistor. As a result, the p-HEI device can activate itself at input power levels less than 5 nW. Using a prototype fabricated in a 0.5- [Formula: see text] bulk CMOS process we validate the functionality of the proposed injector and show that for a fixed input power, its dynamics is quasi-linear with respect to time. The paper also presents measurement results using a cadaver phantom where the fabricated p-HEI device has been integrated with a piezoelectric transducer and is used for self-powered monitoring of mechanical activity.