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Juvenile osteochondritis dissecans (JOCD) is a pediatric disease, which begins with an osteonecrotic lesion in the secondary ossification center which, over time, results in the separation of the necrotic fragment from the parent bone. JOCD predisposes to early-onset osteoarthritis. However, the knowledge gap in JOCD pathomechanisms severely limits current therapeutic strategies. To elucidate its etiology, we conducted a study with induced pluripotent stem cells (iPSCs) from JOCD and control patients. iPSCs from skin biopsies were differentiated to iMSCs (iPSC-derived mesenchymal stromal cells) and subjected to chondrogenic and endochondral ossification, and endoplasmic reticulum (ER)-stress induction assays. Our study, using 3 JOCD donors, showed that JOCD cells have lower chondrogenic capability and their endochondral ossification process differs from control cells; yet, JOCD- and control-cells accomplish osteogenesis of similar quality. Our findings show that endoplasmic reticulum stress sensing and response mechanisms in JOCD cells, which partially regulate chondrocyte and osteoblast differentiation, are related to these differences. We suggest that JOCD cells are more sensitive to ER stress than control cells, and in pathological microenvironments, such as microtrauma and micro-ischemia, JOCD pathogenesis pathways may be initiated. This study is the first, to the best of our knowledge, to realize the important role that resident cells and their differentiating counterparts play in JOCD and to put forth a novel etiological hypothesis that seeks to consolidate and explain previously postulated hypotheses. Furthermore, our results establish well-characterized JOCD-specific iPSC-derived in vitro models and identified potential targets which could be used to improve diagnostic tools and therapeutic strategies in JOCD.

A passively powered wireless sensor based on an inductive-capacitive-resistive (LCR) sensor was developed to measure tensile stress at suture anchors used to repair soft tissue injuries such as ligament and tendon tears. The sensor, which is completely embedded within the suture anchor, consists of a strain gauge mounted on a substrate with a loop connector so a suture can pass through. When the suture experiences a tensile stress, it pulls at the loop connector and the strain gauge, allowing measurement of tensile stress at the suture. In this study, sensors of different designs were fabricated and characterized ex vivo to determine their force sensing capabilities. The effects of sensor placement and orientation on accuracy were also characterized. The LCR suture anchor sensor can be used to assess the quality of soft tissue treatments, such as rotator cuff repairs, as well as to detect failure of the implant post-surgery. The advantages of this sensor are its small size, wireless detection, and battery-free operation, making it ideal for use with both short- and long-term implants.

Successful bone healing in severe trauma depends on early revascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote revascularization following severe bone injuries; however, results have been inconsistent. This is the first study investigating the effects of dual angiogenic growth factors (VEGF and PDGF) with low-dose bone morphogenetic protein-2 (BMP-2; 2.5 µg) on bone healing in a clinically challenging composite bone-muscle injury model. Our hydrogel-based delivery systems demonstrated a more than 90% protein entrapment efficiency and a controlled simultaneous release of three growth factors over 28 days. Co-stimulation of microvascular fragment constructs with VEGF and PDGF promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined VEGF (5 µg) and PDGF (7.5 µg or 15 µg) delivery with a low dose of BMP-2 significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. Notably, the regenerated bone mechanics reached ~60% of intact bone, a value that was previously only achieved by delivery of high-dose BMP-2 (10 µg) in this injury model. Overall, sustained delivery of VEGF, PDFG, and BMP-2 is a promising strategy to promote functional vascularized bone tissue regeneration following severe composite musculoskeletal injury. Although this study is conducted in a clinically relevant composite injury model in rats using a simultaneous release strategy, future studies are necessary to test the regenerative potential of spatiotemporally controlled delivery of triple growth factors on bone healing using large animal models. Statement of significance Volumetric muscle loss combined with delayed union or non-union bone defect causes deleterious effects on bone regeneration even with the supplementation of bone morphogenetic protein-2 (BMP-2). In this study, the controlled delivery of dual angiogenic growth factors (vascular endothelial growth factor [VEGF] + Platelet-derived growth factor [PDGF]) increases vascular growth in vitro. Co-delivering VEGF+PDGF significantly increase the bone formation efficacy of low-dose BMP-2 and improves the mechanics of regenerated bone in a challenging composite bone-muscle injury model.

Magnetoelastic (ME) sensors, which can be remotely activated via magnetic fields, are an excellent choice for wireless monitoring of biological parameters due to their ability to be scaled into different sizes and have their surface functionalized for chemical or biological sensing. In this study, we present the application of a commercially available ME material (Metglas 2826 MB) to develop a sensor system that can monitor the attachment of anchorage-dependent mammalian cells in two-dimensional in vitro cell cultures. Results obtained with the developed sensors and detection system correlated with microscopic image analysis of cell quantification, which showed a linear relationship between the sensor response and attached fibroblast cells on the sensor surface. It was also revealed that the developed ME sensor system is capable of providing temporal profiles of cell growth corresponding to different stages of cell attachment and proliferation in real-time.

Bone nonunions arising from large bone defects and composite injuries remain compelling challenges for orthopedic surgeons. Biological changes associated with nonunions, such as systemic immune dysregulation, can contribute to an adverse healing environment. Bone morphogenetic protein 2 (BMP-2), an osteoinductive and potentially immunomodulatory growth factor, is a promising strategy; however, burst release from the clinical standard collagen sponge delivery vehicle can result in adverse side effects such as heterotopic ossification (HO) and irregular bone structure, especially when using supraphysiological BMP-2 doses for complex injuries at high risk for nonunion. To address this challenge, biomaterials that strongly bind BMP-2, such as heparin methacrylamide microparticles (HMPs), may be used to limit exposure and spatially constrain proteins within the injury site. Here, we investigate moderately high dose BMP-2 delivered in HMPs within an injectable hydrogel system in two challenging nonunion models exhibiting characteristics of systemic immune dysregulation. The HMP delivery system increased total bone volume and decreased peak HO compared to collagen sponge delivery of the same BMP-2 dose. Multivariate analyses of systemic immune markers showed the collagen sponge group correlated with markers that are hallmarks of systemic immune dysregulation, including immunosuppressive myeloid-derived suppressor cells, whereas the HMP groups were associated with immune effector cells, including T cells, and cytokines linked to robust bone regeneration. Overall, our results demonstrate that HMP delivery of moderately high doses of BMP-2 promotes repair of complex bone nonunion injuries and that local delivery strategies for potent growth factors like BMP-2 may positively affect the systemic immune response to traumatic injury.

Osteoarthritis (OA) is a joint degenerative disease that has become one of the leading causes of disability in the world. It is estimated that OA affects 50 million adults in the United States. Currently, there are no FDA-approved treatments that slow OA progression and its treatment is limited to pain management strategies and life style changes. Despite the discovery of several disease-modifying OA drugs (DMOADs) and promising results in preclinical studies, their clinical translation has been significantly limited because of poor intra-articular (IA) bioavailability and challenges in delivering these compounds to tissues of interest within the joint. Here, we review current OA treatments and their effectiveness at reducing joint pain, as well as novel targets for OA treatment and the challenges related to their clinical translation. Moreover, we discuss intra-articular (IA) drug delivery as a promising route of administration, describe its inherent challenges, and review recent advances in biomaterial-based IA drug delivery for OA treatment. Finally, we highlight the potential of tissue targeting in the development of effective IA drug delivery systems.

Intra-articular (IA) injection is an attractive route of administration for the treatment of osteoarthritis (OA). However, free drugs injected into the joint space are rapidly cleared and many of them can induce adverse off-target effects on different IA tissues. To overcome these limitations, we designed nanocomposite 4-arm-poly(ethylene glycol)-maleimide (PEG-4MAL) microgels, presenting cartilage- or synoviocyte-binding peptides, containing poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) as an IA small molecule drug delivery system. Microgels containing rhodamine B (model drug)-loaded PLGA NPs were synthesized using microfluidics technology and exhibited a sustained, near zero-order release of the fluorophore over 16 days in vitro. PEG-4MAL microgels presenting synoviocyte- or cartilage-targeting peptides specifically bound to rabbit and human synoviocytes or to bovine articular cartilage in vitro, respectively. Finally, using a rat model of post-traumatic knee OA, PEG-4MAL microgels were shown to be retained in the joint space for at least 3 weeks without inducing any joint degenerative changes as measured by EPIC-μCT and histology. Additionally, all microgel formulations were found trapped in the synovial membrane and significantly increased the IA retention time of a model small molecule near-infrared (NIR) dye compared to that of the free dye. These results suggest that peptide-functionalized nanocomposite PEG-4MAL microgels represent a promising intra-articular vehicle for tissue-localized drug delivery and prolonged IA drug retention for the treatment of OA.

Mesenchymal stromal cells (MSCs) have failed to consistently demonstrate their therapeutic efficacy in clinical trials, due in part to variability in culture conditions used for their production. Of various culture conditions used for MSC production, aggregate culture has been shown to improve secretory capacity (a putative mechanism of action in vivo) compared with standard monolayer culture. The purpose of this study was to perform multiomics characterization of MSCs cultured in monolayer and as aggregates to identify aspects of cell physiology that differ between these culture conditions to begin to understand cellular-level changes that might be related to secretory capacity. Targeted secretome characterization was performed on multiple batches of MSC-conditioned media, while nontargeted proteome and metabolome characterization was performed and integrated to identify cellular processes differentially regulated between culture conditions. Secretome characterization revealed a reduction in MSC batch variability when cultured as aggregates. Proteome and metabolome characterization showed upregulation of multiple protein and lipid metabolic pathways, downregulation of several cytoskeletal processes, and differential regulation of extracellular matrix synthesis. Integration of proteome and metabolome characterization revealed individual lipid metabolites and vesicle-trafficking proteins as key features for discriminating between culture conditions. Overall, this study identifies several aspects of MSC physiology that are altered by aggregate culture. Further exploration of these processes and pathways is needed to determine their potential role in regulating cell secretory capacity.

Osteoarthritis (OA) is a widespread disease that continues to lack approved and efficacious treatments that modify disease progression. Micronized dehydrated human amnion/chorion membrane (μ-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. The objectives of this study were to characterize the particle size distributions of two μ-dHACM formulations and to investigate the influence of these distributions on the

A healthy musculoskeletal system requires complex functional integration of bone, muscle, cartilage, and connective tissues responsible for bodily support, motion, and the protection of vital organs. Conditions or injuries to musculoskeeltal tissues can devastate an individual's quality of life. Some conditions that are particularly disabling include severe bone and muscle injuries to the extremities and amputations resulting from unmanageable musculoskeletal conditions or injuries. Monitoring and managing musculoskeletal health is intricate because of the complex mechanobiology of these interconnected tissues.For this article, we reviewed literature on implantable biosensors related to clinical data of the musculoskeletal system, therapeutics for complex bone injuries, and osseointegrated prosthetics as example applications.As a result, a brief summary of biosensors technologies is provided along with review of noteworthy biosensors and future developments needed to fully realize the translational benefit of biosensors for musculoskeletal health.Novel implantable biosensors capable of tracking biophysical parameters in vivo are highly relevant to musculoskeletal health because of their ability to collect clinical data relevant to medical decisions, complex trauma treatment, and the performance of osseointegrated prostheses.

Treatment of large bone defects with supraphysiological doses of bone morphogenetic protein-2 (BMP-2) has been associated with complications including heterotopic ossification (HO), inflammation, and pain, presumably due to poor spatiotemporal control of BMP-2. We have previously recapitulated extensive HO in our rat femoral segmental defect model by treatment with high-dose BMP-2 (30 μg). Using this model and BMP-2 dose, our objective was to evaluate the utility of a clinically available human amniotic membrane (AM) around the defect space for guided bone regeneration and reduction of HO. We hypothesized that AM surrounding collagen sponge would attenuate heterotopic ossification compared with collagen sponge alone. In vitro, AM retained more BMP-2 than a synthetic poly(ε-caprolactone) membrane through 21 days. In vivo, as hypothesized, the collagen + AM resulted in significantly less heterotopic ossification and correspondingly, lower total bone volume (BV), compared with collagen sponge alone. Although bone formation within the defect was delayed with AM around the defect, by 12 weeks, defect BVs were equivalent. Torsional stiffness was significantly reduced with AM but was equivalent to that of intact bone. Collagen + AM resulted in the formation of dense fibrous tissue and mineralized tissue, while the collagen group contained primarily mineralized tissue surrounded by marrow-like structures. Especially in conjunction with high doses of growth factor delivered via collagen sponge, these findings suggest AM may be effective as an overlay adjacent to bone healing sites to spatially direct bone regeneration and minimize heterotopic ossification.

Fluid shear stress (FSS) is an important parameter that regulates various cell functions such as proliferation and migration. While there are a number of techniques to generate FSS in vitro, many of them require physical deformation or movement of solid objects to generate the fluid shear, making it difficult to decouple the effects of FSS and mechanical strains. This work describes the development of a non-mechanical means to generate fluid flow and FSS in a 2D in vitro setting. This was accomplished with a magnetohydrodynamic (MHD) pump, which creates liquid flow by generating a Lorentz force through the interaction between an electric field and an orthogonal magnetic field. The MHD pump system presented here consisted of trapezoidal prism-shaped magnets, a pair of platinum electrodes, and a modified petri dish. The system was validated and tested on an in vitro wound model, which is based on analyzing the migration of fibroblast cells through an artificially created scratch on a confluent cell culture surface. Experiments were performed to a control group, an electric field only group, and a group that was subject to fluid flow with the application of both electric field and magnetic field. Results show that fibroblast cells that experienced fluid shear have higher wound closure rate compared to the control group and the electric field only group. The data shows that the MHD pump can be a great tool to study FSS in vitro. Furthermore, due to its fluid flow generation without mechanical force, this system can be adapted and implemented to study the role of FSS and electric field on wound healing in vivo.

Magnetoelastic sensors, typically made of magnetostrictive and magnetically-soft materials, can be fabricated from commercially available materials into a variety of shapes and sizes for their intended applications. Since these sensors are wirelessly interrogated via magnetic fields, they are good candidates for use in both research and industry, where detection of environmental parameters in closed and controlled systems is necessary. Common applications for these sensors include the investigation of physical, chemical, and biological parameters based on changes in mass loading at the sensor surface which affect the sensor’s behavior at resonance. To improve the performance of these sensors, optimization of sensor geometry, size, and detection conditions are critical to increasing their mass sensitivity and detectible range. This work focuses on investigating how the geometry of the sensor influences its resonance spectrum, including the sensor’s shape, size, and aspect ratio. In addition to these factors, heterogeneity in resonance magnitude was mapped for the sensor surface and the effect of the magnetic bias field strength on the resonance spectrum was investigated. Analysis of the results indicates that the shape of the sensor has a strong influence on the emergent resonant modes. Reducing the size of the sensor decreased the sensor’s magnitude of resonance. The aspect ratio of the sensor, along with the bias field strength, was also observed to affect the magnitude of the signal; over or under biasing and aspect ratio extremes were observed to decrease the magnitude of resonance, indicating that these parameters can be optimized for a given shape and size of magnetoelastic sensor.

Volumetric muscle loss (VML) injury is characterized by a non-recoverable loss of muscle fibers due to ablative surgery or severe orthopaedic trauma, that results in chronic functional impairments of the soft tissue. Currently, the effects of VML on the oxidative capacity and adaptability of the remaining injured muscle are unclear. A better understanding of this pathophysiology could significantly shape how VML-injured patients and clinicians approach regenerative medicine and rehabilitation following injury. Herein, the data indicated that VML-injured muscle has diminished mitochondrial content and function (i.e., oxidative capacity), loss of mitochondrial network organization, and attenuated oxidative adaptations to exercise. However, forced PGC-1α over-expression rescued the deficits in oxidative capacity and muscle strength. This implicates physiological activation of PGC1-α as a limiting factor in VML-injured muscle’s adaptive capacity to exercise and provides a mechanistic target for regenerative rehabilitation approaches to address the skeletal muscle dysfunction.

Summary Objective To characterize local disease progression of the medial meniscus transection (MMT) model of post-traumatic osteoarthritis (OA) at the molecular level, in order to establish a baseline for therapeutic testing at the preclinical stage. Design Weight-matched male Lewis rats underwent MMT or sham surgery on the left limb with the right leg as contralateral control. At 1 and 3 weeks post-surgery, tissues were harvested from different areas of the articular cartilage (medial and lateral tibial plateaus, and medial osteophyte region) and synovium (medial and lateral), and analyzed separately. RNA was extracted and used for microarray (RT-PCR) analysis. Results Gene expression changes due to surgery were isolated to the medial side of the joint. Gene changes in chondrocyte phenotype of the medial tibial plateau cartilage preceded changes in tissue composition genes. Differences in inflammatory markers were only observed at the osteophyte region at 3 weeks post-surgery. There was surgical noise in the synovium at week 1, which dissipated at week 3. At this later timepoint, meniscal instability resulted in elevated expression of matrix degradation proteins and osteogenic markers in the synovium and cartilage. Conclusion These results suggest feedback interactions between joint tissues during disease progression. Regional tissue expression differences found in MMT joints indicated similar pathophysiology to human OA, and provided novel insights about this degeneration model. The examination of gene expression at a localized level in multiple tissues provides a well-characterized baseline to evaluate mechanistic effects of potential therapeutic agents on OA disease progression in the MMT model.

Delayed union and nonunion are a significant concern in long bone fractures and spinal fusions. Treatment of nonunion often entails multiple revision surgeries that further increase the financial, physical, and emotional burden on patients. The optimal treatment strategy for nonunions remains unclear in many cases, and the risk of complications after revision procedures remains high. This is in part due to our limited understanding of the biological mechanisms that inhibit proper bone healing and lead to nonunion. And yet, few preclinical models directly investigate how healing is impacted after establishment of nonunion, with most instead primarily focusing on treatment immediately after a fresh bone injury. Here, we utilized a critical size femoral defect model in rats where treatment was delayed 8 weeks post-injury, at which time nonunion was established. In this study, acute and delayed treatments with bone morphogenetic protein-2 (BMP-2) were assessed. We found that delayed treatment resulted in decreased bone formation and reduced mechanical strength compared to acute treatment, even when BMP-2 dose was increased by 2.5 times the acute treatment dose. Interestingly, serum cytokine analysis at 12 weeks post-treatment revealed signs of chronic immune dysregulation after delayed treatment. In particular, non-responders (rats that did not exhibit defect bridging) demonstrated higher overall expression of inflammatory cytokines, including TNFα and IL-1β, compared to responders. These findings suggest that re-establishing long-term immune homeostasis may be critical for successful bone healing, particularly after nonunion. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:299-307, 2019.

Bone morphogenetic protein 2 (BMP-2)-loaded collagen sponges remain the clinical standard for treatment of large bone defects when there is insufficient autograft, despite associated complications. Recent efforts to negate comorbidities have included biomaterials and gene therapy approaches to extend the duration of BMP-2 release and activity. In this study, we compared the collagen sponge clinical standard to chondroitin sulfate glycosaminoglycan (CS-GAG) scaffolds as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) and rhBMP-2 expression via human BMP-2 gene inserted into mesenchymal stem cells (BMP-2 MSC). We demonstrated extended release of rhBMP-2 from CS-GAG scaffolds compared to their collagen sponge counterparts, and further extended release from CS-GAG gels seeded with BMP-2 MSC. When used to treat a challenging critically sized femoral defect model in rats, both rhBMP-2 and BMP-2 MSC in CS-GAG induced comparable bone formation to the rhBMP-2 in collagen sponge, as measured by bone volume, strength, and stiffness. We conclude that CS-GAG scaffolds are a promising delivery vehicle for controlling the release of rhBMP-2 and to mediate the repair of critically sized segmental bone defects. Stem Cells Translational Medicine 2019;8:575–585

The etiology of joint tissue degeneration following rotator cuff tear remains unclear. Thus, the purpose of this study was to understand the timeline of protease activity in the soft tissues of the shoulder (tendon, muscle, and cartilage) that may lead to down-stream degeneration following rotator cuff tear. A well-established rat model involving suprascapular nerve denervation and supraspinatus/infraspinatus tendon transection was employed. Histological staining and/or micro-computed tomography (µCT) were used to observe structural damage in the supraspinatus tendon and muscle, humeral head cartilage, and subchondral bone. Multiplex gelatin zymography was utilized to assess protease activity in the supraspinatus tendon and muscle, and humeral head cartilage. Zymography analysis demonstrated that cathepsins were upregulated in the first week in all tissues, while MMP-2 maintained prolonged activity in supraspinatus tendon between 1 and 3 weeks and increased only at 3 weeks in supraspinatus muscle. In supraspinatus tendon, increased cathepsin L and MMP-2 activity in the first week was concurrent with matrix disorganization and infiltration of inflammatory cells. In contrast, significant upregulation of cathepsin L and K activity in supraspinatus muscle and humeral head cartilage did not correspond to any visible tissue damage at 1 week. However, focal defects developed in half of all animals' humeral head cartilage by 12 weeks (volume: 0.12 ± 0.09 mm3 ). This work provides a more comprehensive understanding of biochemical changes to joint tissue over time following rotator cuff tear. Overall, this provides insight into potential therapeutic targets and will better inform ideal intervention times and treatments for each tissue. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:490-502, 2019.

Polyether-ether-ketone (PEEK) is one of the most common materials used for load-bearing orthopaedic devices due to its radiolucency and favorable mechanical properties. However, current smooth-surfaced PEEK implants can lead to fibrous encapsulation and poor osseointegration. This study compared the in vitro and in vivo bone response to two smooth PEEK alternatives: porous PEEK and plasma-sprayed titanium coatings on PEEK. MC3T3 cells were grown on smooth PEEK, porous PEEK, and Ti-coated PEEK for 14 days and assayed for calcium content, osteocalcin, VEGF and ALP activity. Osseointegration was investigated by implanting cylindrical implants into the proximal tibiae of male Sprague Dawley rats for 8 weeks. Bone-implant interfaces were evaluated using μCT, histology and pullout testing. Cells on porous PEEK surfaces produced more calcium, osteocalcin, and VEGF than smooth PEEK and Ti-coated PEEK groups. Bone ingrowth into porous PEEK surfaces was comparable to previously reported porous materials and correlated well between μCT and histology analysis. Porous PEEK implants exhibited greater pullout force, stiffness and energy-to-failure compared to smooth PEEK and Ti-coated PEEK, despite Ti-coated PEEK exhibiting a high degree of bone-implant contact. These results are attributed to increased mechanical interlocking of bone with the porous PEEK implant surface. Overall, porous PEEK was associated with improved osteogenic differentiation in vitro and greater implant fixation in vivo compared to smooth PEEK and Ti-coated PEEK. These results suggest that not all PEEK implants inherently generate a fibrous response and that topography has a central role in determining implant osseointegration.

Severe traumatic injuries are a widespread and challenging clinical problem, and yet the factors that drive successful healing and restoration of function are still not well understood. One recently identified risk factor for poor healing outcomes is a dysregulated immune response following injury. In a preclinical model of orthopedic trauma, we demonstrate that distinct systemic immune profiles are correlated with impaired bone regeneration. Most notably, elevated blood levels of myeloid-derived suppressor cells (MDSCs) and the immunosuppressive cytokine interleukin-10 (IL-10) are negatively correlated with functional bone regeneration as early as 1 wk posttreatment. Nonlinear multivariate regression also implicated these two factors as the most influential in predictive computational models. These results support a significant relationship between early systemic immune responses to trauma and subsequent local bone regeneration and indicate that elevated circulating levels of MDSCs and IL-10 may be predictive of poor functional healing outcomes and represent novel targets for immunotherapeutic intervention.

Purpose: Mechanical loading of bone defects through rehabilitation is a promising approach to stimulate repair and reduce nonunion risk; however, little is known about how therapeutic mechanical stimuli modulate early-stage repair before mineralized bone formation. The objective of this study was to investigate the early effects of osteogenic loading on cytokine expression and angiogenesis during the first 3 weeks of BMP-2 mediated segmental bone defect repair. Materials and Methods: A rat model of BMP-2 mediated bone defect repair was subjected to an osteogenic mechanical loading protocol using ambulatory rehabilitation and a compliant, load-sharing fixator with an integrated implantable strain sensor. The effect of fixator load-sharing on local tissue strain, angiogenesis, and cytokine expression was evaluated. Results: Using sensor readings for local measurements of boundary conditions, finite element simulations showed strain became amplified in remaining soft tissue regions between 1 and 3 weeks (Week 3: load-sharing: ���1.89 �� 0.35% and load-shielded: ���1.38 �� 0.35% vs. Week 1: load-sharing: ���1.54 �� 0.17%; load-shielded: ���0.76 �� 0.06%). Multivariate analysis of cytokine arrays revealed that load-sharing significantly altered expression profiles in the defect tissue at 2 weeks compared to load-shielded defects. Specifically, loading reduced VEGF (p = 0.052) and increased CXCL5 (LIX) levels. Subsequently, vascular volume in loaded defects was reduced relative to load-shielded defects but similar to intact bone at 3 weeks. Endochondral bone repair was also observed histologically in loaded defects at 3 weeks. Conclusions: Together, these results demonstrate that moderate ambulatory strains previously shown to stimulate bone regeneration significantly alter early angiogenic and cytokine signaling and may promote endochondral ossification.

Extracellular matrix deformation temporally regulates angiogenesis and upstream angiogenic and mechanosensitive pathways., Mechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Previously, we found that dynamic matrix compression in vivo potently regulated revascularization during bone tissue regeneration; however, whether matrix deformations directly regulate angiogenesis remained unknown. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Immediate load initiation inhibited angiogenesis and expression of early sprout tip cell selection genes, while delayed loading enhanced microvascular network formation and upstream signaling pathways. This research provides foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

The objective of this study was to investigate the controlled release of two growth factors (BMP-2 and VEGF) as a treatment strategy for clinically challenging composite injuries, consisting of a segmental bone defect and volumetric muscle loss. This is the first investigation of dual growth factor delivery in a composite injury model using an injectable smart delivery system consisting of heparin microparticles and alginate gel. The loading efficiency of growth factors into these biomaterials was found to be >90%, revealing a strong affinity of VEGF and BMP-2 to heparin and alginate. The system could achieve simultaneous or sequential release of VEGF and BMP-2 by varying the loading strategy. Single growth factor delivery (VEGF or BMP-2 alone) significantly enhanced vascular growth in vitro. However, no synergistic effect was observed for dual growth factor (BMP-2 + VEGF) delivery. Effective bone healing was achieved in all treatment groups (BMP-2, simultaneous or sequential delivery of BMP-2 and VEGF) in the composite injury model. The mechanics of the regenerated bone reached a maximum strength of ∼52% of intact bone with sequential delivery of VEGF and BMP-2. Overall, simultaneous or sequential co-delivery of low-dose BMP-2 and VEGF failed to fully restore the mechanics of bone in this injury model. Given the severity of the composite injury, VEGF alone may not be sufficient to establish mature and stable blood vessels when compared with previous studies co-delivering BMP-2+VEGF enhanced bone tissue regeneration. Hence, future studies are warranted to develop an alternative treatment strategy focusing on better control over growth factor dose, spatiotemporal delivery, and additional growth factors to regenerate fully functional bone tissue.HighlightsWe developed a smart growth factor delivery system using heparin microparticles and alginate that facilitates tunable delivery of VEGF and BMP-2 in a simultaneous or sequential manner by merely varying the loading strategy.In vitro, both VEGF and BMP-2 alone promoted vascular growth; however, VEGF was significantly more potent, and there was no detectable benefit of co-delivery.In vivo, both BMP-2 alone and co-delivery of VEGF and BMP-2 promoted bone formation in the challenging bone/muscle polytrauma model; however, none of the treatment groups restored biomechanical properties to that of uninjured bone.

Biomaterial scaffolds have served as the foundation of tissue engineering and regenerative medicine. However, scaffold systems are often difficult to scale in size or shape in order to fit defect-specific dimensions, and thus provide only limited spatiotemporal control of therapeutic delivery and host tissue responses. Here, a lithography-based three-dimensional (3D) printing strategy is used to fabricate a novel miniaturized modular LEGO-like cage scaffold system, which can be assembled and scaled manually with ease. Scalability is based on an intuitive concept of stacking modules, like conventional LEGO blocks, allowing for literally thousands of potential geometric configurations, and without the need for specialized equipment. Moreover, the modular hollow-cage design allows each unit to be loaded with biologic cargo of different compositions, thus enabling controllable and easy patterning of therapeutics within the material in 3D. In summary, the concept of miniaturized cage designs with such straight-forward assembly and scalability, as well as controllable loading properties, is a flexible platform that can be extended to a wide range of materials for improved biological performance.TOC3D printed LEGO-like hollow microcages can be easily assembled, adjoined, and stacked-up to suit the complexity of defect tissues; aid spatial loading of cells and biomolecules; instruct cells migration three-dimensionally; and facilitate cell invasion and neovascularization in-vivo, thus accelerating the process of tissue healing and new tissue formation.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:485-502, 2020.

Neovascularization is a critical early step toward successful tissue regeneration during wound healing. While vasculature has long been recognized as highly mechanosensitive (to fluid shear, pulsatile luminal pressure, etc.), the effects of extracellular matrix strains on angiogenesis are poorly understood. Previously, we found that dynamic matrix compression in vivo potently regulated neovascular growth during tissue regeneration; however, whether matrix deformations directly regulate00 angiogenesis remained unknown. Here, we tested the effects of load initiation time, strain magnitude, and mode of compressive deformation (uniform compression vs. compressive indentation that also introduced shear stress) on neovascularization and key angiogenic and mechanotransduction signaling pathways by microvascular fragments in vitro. We hypothesized that neovascularization would be enhanced by delayed, moderate compression and inhibited by early, high magnitude compression and by compressive indentation. Consistent with our hypothesis, early, high magnitude loading inhibited vessel growth, while delayed loading enhanced vessel growth. Compressive indentation led to longer, more branched networks than uniform compression – particularly at high strain magnitude. Gene expression was differentially regulated by time of load initiation; genes associated with active angiogenic sprouts were downregulated by early loading but upregulated by delayed loading. Canonical gene targets of the YAP/TAZ mechanotransduction pathway were increased by loading and abrogated by pharmacological YAP inhibition. Together, these data demonstrate that neovascularization is directly responsive to dynamic matrix strain and is particularly sensitive to the timing of load initiation. This work further identifies putative mechanoregulatory angiogenic mechanisms and implicates a critical role for dynamic mechanical cues in vascularized tissue regeneration.Statement of SignificanceMechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, remarkably little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Across all tested magnitudes and modes, microvascular network formation and upstream signaling were powerfully regulated by the timing of load initiation. This work provides a new foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

Orthopedic biomaterial-associated infections remain a large clinical challenge, particularly with open fractures and segmental bone loss. Invasion and colonization of bacteria within immune-privileged canalicular networks of the bone can lead to local, indolent infections that can persist for years without symptoms before eventual catastrophic hardware failure. Host immunity is essential for bacterial clearance and an appropriate healing response, and recent evidence has suggested an association between orthopedic trauma and systemic immune dysregulation and immunosuppression. However, the impact of a local infection on this systemic immune response and subsequent effects on the local response is poorly understood and has not been a major focus for addressing orthopedic injuries and infections. Therefore, this study utilized a model of orthopedic biomaterial-associated infection to investigate the effects of infection on the long-term immune response. Here, despite persistence of a local, indolent infection lacking outward symptoms, there was still evidence of long-term immune dysregulation with systemic increases in MDSCs and decreases in T cells compared to non-infected trauma. Further, the trauma only group exhibited a regulated and coordinated systemic cytokine response, which was not present in the infected trauma group. Locally, the infection group had attenuated macrophage infiltration in the local soft tissue compared to the non-infected group. Our results demonstrate widespread impacts of a localized orthopedic infection on the systemic and local immune responses. Characterization of the immune response to orthopedic biomaterial-associated infection may identify key targets for immunotherapies that could optimize both regenerative and antibiotic interventions, ultimately improving outcomes for these patients.

Heparin-based microparticles improve spatial localization of BMP-2 delivery, decreasing bone formation outside of the injury site., Supraphysiologic doses of bone morphogenetic protein-2 (BMP-2) are used clinically to promote bone formation in fracture nonunions, large bone defects, and spinal fusion. However, abnormal bone formation (i.e., heterotopic ossification) caused by rapid BMP-2 release from conventional collagen sponge scaffolds is a serious complication. We leveraged the strong affinity interactions between heparin microparticles (HMPs) and BMP-2 to improve protein delivery to bone defects. We first developed a computational model to investigate BMP-2–HMP interactions and demonstrated improved in vivo BMP-2 retention using HMPs. We then evaluated BMP-2–loaded HMPs as a treatment strategy for healing critically sized femoral defects in a rat model that displays heterotopic ossification with clinical BMP-2 doses (0.12 mg/kg body weight). HMPs increased BMP-2 retention in vivo, improving spatial localization of bone formation in large bone defects and reducing heterotopic ossification. Thus, HMPs provide a promising opportunity to improve the safety profile of scaffold-based BMP-2 delivery.

Type III fractures typically involve segmental bone loss with extensive adjacent soft tissue injury to muscle and vasculature. Such severe composite injuries to bone and muscle often require multiple treatment procedures and are associated with significantly higher rates of complications, including non-union, infection, prolonged disability, and amputation. Successful bone healing depends on early re-vascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote re-vascularization following severe bone injuries, however results have been inconsistent. In this study, co-stimulation of microvascular fragment constructs with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined delivery of VEGF and PDGF with a low dose of bone morphogenetic protein-2 (BMP-2) significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. This study demonstrates the potential for a combined osteoinductive and angiogenic growth factor delivery strategy to promote functional bone regeneration following severe composite musculoskeletal injury.

Large-scale manufacturing of therapeutic cells requires bioreactor technologies with online feedback control enabled by monitoring of secreted biomolecular critical quality attributes (CQAs). Electrospray ionization mass spectrometry (ESI-MS) is a highly sensitive label-free method to detect and identify biomolecules, but requires extensive sample preparation before analysis, making online application of ESI-MS challenging. We present a microfabricated, monolithically integrated device capable of continuous sample collection, treatment, and direct infusion for ESI-MS detection of biomolecules in high-salt solutions. The dynamic mass spectrometry probe (DMSP) uses a microfluidic mass exchanger to rapidly condition samples for online MS analysis by removing interfering salts, while concurrently introducing MS signal enhancers to the sample for sensitive biomolecular detection. Exploiting this active conditioning capability increases MS signal intensity and signal-to-noise ratio. As a result, sensitivity for low-concentration biomolecules is significantly improved, and multiple proteins can be detected from chemically complex samples. Thus, the DMSP has significant potential to serve as an enabling portion of a novel analytical tool for discovery and monitoring of CQAs relevant to therapeutic cell manufacturing.

Severe musculoskeletal trauma is one of the most prevalent types of trauma in both combat-wounded and civilian patients. However, despite advances in trauma care management, mortality and complication rates remain high. Recently, systemic immune dysregulation and immunosuppression has been implicated as a main contributor to unsatisfactory success of intervention strategies and poor outcomes in a subset of trauma patients. The importance of the immune system in wound healing and tissue regeneration suggests that for optimal success of regenerative medicine strategies, this underlying systemic immune dysregulation must be addressed, highlighting a strong need for immunomodulatory approaches to augment regenerative therapies. This review addresses what is currently known about systemic immune dysregulation following severe trauma, previous attempts at immunomodulation, considerations for the development of future immunomodulatory strategies, and animal models for testing these therapeutics. The severity of immune dysregulation over time, both systemically and locally, is impacted by injury severity and patient-specific factors, such as age and co-morbidities. Varying degrees of systemic immune dysregulation may require different and personalized approaches to restore immune homeostasis. Further, the timing of treatment and the site of delivery must be considered to determine the most safe and effective intervention strategy. In order to test and develop these immunomodulatory therapeutics aimed at improving outcomes of regenerative interventions, robust pre-clinical models are needed that recapitulate systemic immune dysregulation and clinical scenarios. Overall, more fundamental research is needed to better understand the complex interactions between the musculoskeletal system and the immune system, as well as the importance of systemic and local immune function on healing. Ultimately, this information could lead to new immunomodulatory strategies to enhance regeneration for severe musculoskeletal trauma patients.

Mechanical loads exerted on the skeleton during activities such as walking are important regulators of bone repair, but dynamic biomechanical signals are difficult to measure inside the body. The inability to measure the mechanical environment in injured tissues is a significant barrier to developing integrative regenerative and rehabilitative strategies that can accelerate recovery from fracture, segmental bone loss, and spinal fusion. Here we engineered an implantable strain sensor platform and longitudinally measured strain across a bone defect in real-time throughout rehabilitation. The results showed that load-sharing permitted by a load-sharing fixator initially delivered a two-fold increase in deformation magnitude, subsequently increased mineralized bridging by nearly three-fold, and increased bone formation by over 60%. These data implicate a critical role for early mechanical cues on the long term healing response as strain cycle magnitude at 1 week (before appreciable healing occurred) had a significant positive correlation with the long-term bone regeneration outcomes. Furthermore, we found that sensor readings correlated with the status of healing, suggesting a role for strain sensing as an X-ray-free healing assessment platform. Therefore, non-invasive strain measurements may possess diagnostic potential to evaluate bone repair and reduce clinical reliance on current radiation-emitting imaging methods. Together, this study demonstrates a promising framework to quantitatively develop and exploit mechanical rehabilitation strategies that enhance bone repair.

Duchenne muscular dystrophy is a severe muscle wasting disease due to the absence of the dystrophin protein from the muscle cell membrane, which renders the muscle susceptible to continuous damage. In Duchenne muscular dystrophy patients, muscle weakness, together with cycles of degeneration/regeneration and replacement with noncontractile tissue, limit mobility and lifespan. Because the loss of dystrophin results in loss of polarity and a reduction in the number of self-renewing satellite cells, it is postulated that these patients could achieve an improved quality of life if delivered cells could restore satellite cell function. In this study, we used both an established myotoxic injury model in wild-type (WT) mice and mdx mice alone (spontaneous muscle damage). Single (SC) and aggregated (AGG) mesenchymal stem cells (MSCs) were injected into the gastrocnemius muscles 4 hr after injury (WT mice). The recovery of peak isometric torque was longitudinally assessed over 5 weeks, with earlier takedowns for histological assessment of healing (fibre cross-section area and central nucleation) and MSC retention. AGG-treated WT mice had significantly greater torque recovery at Day 14 than SC or saline-treated mice and a greater CSA at Day 10, compared with SC/saline. AGG-treated mdx mice had a greater peak isometric torque compared with SC/saline. In vitro immunomodulatory factor secretion of AGG-MSCs was higher than SC-MSCs for all tested growth factors with the largest difference observed in hepatocyte growth factor. Future studies are necessary to pair immunomodulatory factor secretion with functional attributes, to better predict the potential therapeutic value of MSC treatment modalities.

Significance Orthopedic implant infections require long-term antibiotic therapy and surgical debridement to successfully retain the implant; however, therapeutic failure can lead to implant removal. Here an injectable PEG-based hydrogel that adheres to exposed tissue and fracture surfaces is engineered to deliver the antimicrobial enzyme lysostaphin to infected, implant-fixed, mouse femoral fractures. Lysostaphin encapsulation within the hydrogel enhances enzyme stability while providing enhanced antibiofilm activity and serving as a controlled delivery platform. In a preclinical animal model of orthopedic-implant infection, we show that lysostaphin-delivering hydrogels outperform prophylactic antibiotic therapy and soluble lysostaphin, by eradicating infection while promoting bone repair. Importantly, lysostaphin-delivering hydrogels are effective against antibiotic-resistant infections. This lysostaphin delivery platform could be highly effective at treating and preventing implant infections., Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair.

Implantable wireless sensors have been used for real-time monitoring of chemicals and physical conditions of bones, tendons and muscles to diagnose and study orthopedic diseases and injuries. Due to the importance of these sensors in orthopedic care, a critical review, which not only analyzes the underlying technologies but also their clinical implementations and challenges, will provide a landscape view on their current state and their future clinical role.By conducting an extensive literature search and following the leaders of orthopedic implantable wireless sensors, this review covers the battery-powered and battery-free wireless implantable sensor technologies, and describes their implementation for hips, knees, spine, and shoulder stress/strain monitoring. Their advantages, limitations, and clinical challenges are also described.Currently, implantable wireless sensors are mostly limited for scientific investigations and demonstrative experiments. Although rapid advancement in sensors and wireless technologies will push the reliability and practicality of these sensors for clinical realization, regulatory constraints and financial viability in medical device industry may curtail their continuous adoption for clinical orthopedic applications. In the next five years, these sensors are expected to gain increased interest from researchers, but wide clinical adoption is still unlikely.

As a potential treatment for osteoarthritis (OA), we have developed injectable and hydrolytically degradable heparin-based biomaterials with tunable sulfation for the intra-articular delivery of tumor necrosis factor-alpha stimulated gene-6 (TSG-6), a protein known to inhibit plasmin which may degrade extracellular matrix within OA joints. We first assessed the effect of heparin sulfation on TSG-6 anti-plasmin activity and found that while fully sulfated (Hep) and heparin desulfated at only the N position (Hep-N) significantly enhanced TSG-6 bioactivity in vitro, fully desulfated heparin (Hep-) had no effect, indicating that heparin sulfation plays a significant role in modulating TSG-6 bioactivity. Next, TSG-6 loaded, degradable 10 wt% Hep-N microparticles (MPs) were delivered via intra-articular injection into the knee at 1, 7, and 15 days following medial meniscal transection (MMT) injury in a rat model. After 21 days, cartilage thickness, volume, and attenuation were significantly increased with soluble TSG-6, indicating degenerative changes. In contrast, no significant differences were observed with TSG-6 loaded MP treatment, demonstrating that TSG-6 loaded MPs reduced cartilage damage following MMT injury. Ultimately, our results indicate that Hep-N can enhance TSG-6 anti-plasmin activity and that Hep-N-based biomaterials may be an effective method for TSG-6 delivery to treat OA.

Summary Abaloparatide, a novel PTH1 receptor agonist, increased bone formation in osteopenic ovariectomized cynomolgus monkeys while increasing cortical and trabecular bone mass. Abaloparatide increased bone strength and maintained or enhanced bone mass-strength relationships, indicating preserved or improved bone quality. Introduction Abaloparatide is a selective PTH1R activator that is approved for the treatment of postmenopausal osteoporosis. The effects of 16 months of abaloparatide administration on bone formation, resorption, density, and strength were assessed in adult ovariectomized (OVX) cynomolgus monkeys (cynos). Methods Sixty-five 9–18-year-old female cynos underwent OVX surgery, and 15 similar cynos underwent sham surgery. After a 9-month period without treatments, OVX cynos were allocated to four groups that received 16 months of daily s.c. injections with either vehicle (n = 17) or abaloparatide (0.2, 1, or 5 μg/kg/day; n = 16/dose level), while Sham controls received s.c. vehicle (n = 15). Bone densitometry (DXA, pQCT, micro-CT), qualitative bone histology, serum calcium, bone turnover markers, bone histomorphometry, and bone strength were among the key measures assessed. Results At the end of the 9-month post-surgical bone depletion period, just prior to the treatment phase, the OVX groups exhibited increased bone turnover markers and decreased bone mass compared with sham controls. Abaloparatide administration to OVX cynos led to increased bone formation parameters, including serum P1NP and endocortical bone formation rate. Abaloparatide administration did not influence serum calcium levels, bone resorption markers, cortical porosity, or eroded surfaces. Abaloparatide increased bone mass at the whole body, lumbar spine, tibial diaphysis, femoral neck, and femoral trochanter. Abaloparatide administration was associated with greater lumbar vertebral strength, and had no adverse effects on bone mass-strength relationships for the vertebrae, femoral neck, femoral diaphysis, or humeral cortical beams. Conclusions Abaloparatide administration was associated with increases in bone formation, bone mass and bone strength, and with maintenance of bone quality in OVX cynos, without increases in serum calcium or bone resorption parameters. Electronic supplementary material The online version of this article (10.1007/s00198-017-4323-6) contains supplementary material, which is available to authorized users.