A multi-institutional study of short-term mortality in COVID-positive patients undergoing hip fracture surgery: is survival better than expected?

PURPOSE: Early reports of 30-day mortality in COVID-positive patients with hip fracture were often over 30% and were higher than historical rates of 10% in pre-COVID studies. We conducted a multi-institutional retrospective cohort study to determine whether the incidence of 30-day mortality and complications in COVID-positive patients undergoing hip fracture surgery is as high as initially reported. METHODS: A retrospective chart review was performed at 11 level I trauma centers from January 1, 2020 to May 1, 2022. Patients 50 years or older undergoing hip fracture surgery with a positive COVID test at the time of surgery were included. The primary outcome measurements were the incidence of 30-day mortality and complications. Post-operative outcomes were reported using proportions with 95% confidence interval (C.I.). RESULTS: Forty patients with a median age of 71.5 years (interquartile range, 50-87 years) met the criteria. Within 30-days, four patients (10%; 95% C.I. 3-24%) died, four developed pneumonia, three developed thromboembolism, and three remained intubated post-operatively. Increased age was a statistically significant predictor of 30-day mortality (p = 0.01), with all deaths occurring in patients over 80 years. CONCLUSION: In this multi-institutional analysis of COVID-positive patients undergoing hip fracture surgery, 30-day mortality was 10%. The 95% C.I. did not include 30%, suggesting that survival may be better than initially reported. While COVID-positive patients with hip fractures have high short-term mortality, the clinical situation may not be as dire as initially described, which may reflect initial publication bias, selection bias introduced by testing, or other issues. LEVELS OF EVIDENCE: Therapeutic Level III.

Biomechanical Stimulation of Muscle Constructs Influences Phenotype of Bone Constructs by Modulating Myokine Secretion.

Diabetes is a chronic metabolic disorder that can lead to diabetic myopathy and bone diseases. The etiology of musculoskeletal complications in such metabolic disorders and the interplay between the muscular and osseous systems are not well understood. Exercise training promises to prevent diabetic myopathy and bone disease and offer protection. Although the muscle-bone interaction is largely biomechanical, the muscle secretome has significant implications for bone biology. Uncoupling effects of biophysical and biochemical stimuli on the adaptive response of bone during exercise training may offer therapeutic targets for diabetic bone disease. Here, we have developed an in vitro model to elucidate the effects of mechanical strain on myokine secretion and its impact on bone metabolism decoupled from physical stimuli. We developed bone constructs using cross-linked gelatin, which facilitated osteogenic differentiation of osteoprogenitor cells. Then muscle constructs were made from fibrin, which enabled myoblast differentiation and myotube formation. We investigated the myokine expression by muscle constructs under strain regimens replicating endurance (END) and high-intensity interval training (HIIT) in hyperglycemic conditions. In monocultures, both regimens induced higher expression of Il15 and Igf1, whereas END supported more myoblast differentiation and myotube maturation than HIIT. When co-cultured with bone constructs, HIIT regimen increased Glut4 expression in muscle constructs more than END, supporting higher glucose uptake. Likewise, the muscle constructs under the HIIT regimen promoted a healthier and more matured bone phenotype than END. Under static conditions, myostatin (Mstn) expression was significantly downregulated in muscle constructs co-cultured with bone constructs compared with monocultures. Together, our in vitro co-culture system allowed orthogonal manipulation of mechanical strain on muscle constructs while facilitating bone-muscle biochemical cross-talk. Such systems can provide an individualized microenvironment that allows decoupled biomechanical manipulation, help identify molecular targets, and develop engineered therapies for metabolic bone disease. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Carbonized Tetracycline: a new class of nanomaterial with tuneable antioxidant, reduced cytotoxicity, immunomodulatory, and osteogenic properties.

Tetracycline (TET), a broad-spectrum antibiotic, also possesses different non-antibiotic activities such as inhibition of metalloproteinase (MMP), anti-inflammatory, antioxidant, high bone affinity, etc. However, the comparatively low efficacy of these non-antibiotic properties along with adverse effects such as hyperpigmentation, phototoxicity, long-term skeletal retention, etc. have not helped their broad utilization similar to their use as an antibiotic. In a unique attempt to improve the non-antibiotic properties while reducing the adverse effects, we converted the TET to nano-carbons through partial carbonization. After sorting out two water-dispersible C-TETs (C-TET HT - hydrothermal and C-TET HP - hot plate) based on their improved antioxidant activity, they have been characterized through a host of analytical techniques that showed distinct differences in morphology, size, shape, and surface functionality. Excitingly, the C-TET HT and C-TET HP have shown differential biological activity in a dosage and time-dependent manner in terms of cytotoxicity, immunomodulation, and osteogenic activity that was found to be associated with their carbonized parameters. Overall, the carbonized nano-drugs, C-TET HT and C-TET HP have presented substantial early promises on their non-antibiotic properties that could be further explored to develop into some effective therapeutics.

A Murine Delayed-Healing Model Associates Immune Response with Functional Bone Regeneration after Trauma.

Nonunion and delayed-union fractures pose a significant clinical challenge, often leading to prolonged morbidity and impaired quality of life. Fracture-induced hematoma and acute inflammation are crucial for establishing the healing cascade. However, aberrant inflammatory phenotypes can suppress healing and cause bone resorption. Elucidating these mechanisms is necessary to develop potent immunomodulatory therapies and prevent nonunion. Here, we report a delayed fracture healing model enabling the modulation of interfragmentary strain that mimics the etiology of hypertrophic nonunions to elucidate the role of dysregulated immune response in poor healing outcomes. High interfragmentary strain (>15%) was associated with larger callus volumes but delayed bone healing, increased inflammation, and inferior healing outcomes, while lower strain levels (<5%) corresponded to normal bone healing. In addition, we found distinct differences in the ossification, chondrification, and fibrosis patterns between high and low-strain groups, underscoring the significant impact of strain on the healing process. A comprehensive analysis of the systemic immune response revealed dynamic alterations in immune cell populations and factors, particularly within the early hours and days post-fracture. Several immune factors exhibited significant correlations with various functional healing outcomes, indicating their potential as predictive markers for assessing fracture healing progression. Our results also highlighted the significance of timely resolution of proinflammatory signals and the elevation of pro-regenerative immune cell phenotypes in promoting bone regeneration. Multivariate analysis revealed that CD25+ T-regulatory cells were influential in predicting proper bone healing, followed by CD206+ macrophages, underscoring the pivotal role of immune cell populations in the bone healing process. In conclusion, our study provides valuable insights into the intricate interplay between interfragmentary strain, immune response, and the ultimate outcomes of fracture healing. By shedding light on the underlying mechanisms that drive hypertrophic nonunion pathogenesis, our research lays the foundation for enhanced surgical management of nonunions and offers a promising avenue for developing targeted therapeutic interventions and personalized treatment strategies for individuals suffering from fracture nonunion.

GDF8 inhibition enhances musculoskeletal recovery and mitigates posttraumatic osteoarthritis following joint injury.

Musculoskeletal disorders contribute substantially to worldwide disability. Anterior cruciate ligament (ACL) tears result in unresolved muscle weakness and posttraumatic osteoarthritis (PTOA). Growth differentiation factor 8 (GDF8) has been implicated in the pathogenesis of musculoskeletal degeneration following ACL injury. We investigated GDF8 levels in ACL-injured human skeletal muscle and serum and tested a humanized monoclonal GDF8 antibody against a placebo in a mouse model of PTOA (surgically induced ACL tear). In patients, muscle GDF8 was predictive of atrophy, weakness, and periarticular bone loss 6 months following surgical ACL reconstruction. In mice, GDF8 antibody administration substantially mitigated muscle atrophy, weakness, and fibrosis. GDF8 antibody treatment rescued the skeletal muscle and articular cartilage transcriptomic response to ACL injury and attenuated PTOA severity and deficits in periarticular bone microarchitecture. Furthermore, GDF8 genetic deletion neutralized musculoskeletal deficits in response to ACL injury. Our findings support an opportunity for rapid targeting of GDF8 to enhance functional musculoskeletal recovery and mitigate the severity of PTOA after injury.

Magnetic nanocomplexes coupled with an external magnetic field modulate macrophage phenotype - a non-invasive strategy for bone regeneration.

Chronic inflammation is a major cause for the pathogenesis of musculoskeletal diseases such as fragility fracture, and nonunion. Studies have shown that modulating the immune phenotype of macrophages from proinflammatory to prohealing mode can heal recalcitrant bone defects. Current therapeutic strategies predominantly apply biochemical cues, which often lack target specificity and controlling their release kinetics in vivo is challenging spatially and temporally. We show a magnetic iron-oxide nanocomplexes (MNC)-based strategy to resolve chronic inflammation in the context of promoting fracture healing. MNC internalized pro-inflammatory macrophages, when coupled with an external magnetic field, exert an intracellular magnetic force on the cytoskeleton, which promotes a prohealing phenotype switch. Mechanistically, the intracellular magnetic force perturbs actin polymerization, thereby significantly reducing nuclear to cytoplasm redistribution of MRTF-A and HDAC3, major drivers of inflammatory and osteogenic gene expressions. This significantly reduces Nos2 gene expression and subsequently downregulates the inflammatory response, as confirmed by quantitative PCR analysis. These findings are a proof of concept to develop MNC-based resolution-centric therapeutic intervention to direct macrophage phenotype and function towards healing and can be translated either to supplement or replace the currently used anti-inflammatory therapies for fracture healing.

Licensing microgels prolong the immunomodulatory phenotype of mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) are sensors of inflammation, and they exert immunomodulatory properties through the secretion of cytokines and exosomes and direct cell-cell interactions. MSC are routinely used in clinical trials and effectively resolve inflammatory conditions. Nevertheless, inconsistent clinical outcomes necessitate the need for more robust therapeutic phenotypes. The immunomodulatory properties of MSC can be enhanced and protracted by priming (aka licensing) them with IFNγ and TNFα. Yet these enhanced properties rapidly diminish, and prolonged stimulation could tolerize their response. Hence a balanced approach is needed to enhance the therapeutic potential of the MSC for consistent clinical performance. Here, we investigated the concentration-dependent effects of IFNγ and TNFα and developed gelatin-based microgels to sustain a licensed MSC phenotype. We show that IFNγ treatment is more beneficial than TNFα in promoting an immunomodulatory MSC phenotype. We also show that the microgels possess integrin-binding sites to support adipose tissue-derived MSC (AD-MSC) attachment and a net positive charge to sequester the licensing cytokines electrostatically. Microgels are enzymatically degradable, and the rate is dependent on the enzyme concentration and matrix density. Our studies show that one milligram of microgels by dry mass can sequester up to 641 ± 81 ng of IFNγ. Upon enzymatic degradation, microgels exhibited a sustained release of IFNγ that linearly correlated with their degradation rate. The AD-MSC cultured on the IFNγ sequestered microgels displayed efficient licensing potential comparable to or exceeding the effects of bolus IFNγ treatment. When cultured with proinflammatory M1-like macrophages, the AD-MSC-seeded on licensing microgel showed an enhanced immunomodulatory potential compared to untreated AD-MSC and AD-MSC treated with bolus IFNγ treatment. Specifically, the AD-MSC seeded on licensing microgels significantly upregulated Arg1, Mrc1, and Igf1, and downregulated Tnfα in M1-like macrophages compared to other treatment conditions. These licensing microgels are a potent immunomodulatory approach that shows substantial promise in elevating the efficacy of current MSC therapies and may find utility in treating chronic inflammatory conditions.

Injectable nanoporous microgels generate vascularized constructs and support bone regeneration in critical-sized defects.

Large and aberrant bone fractures require ossification and concomitant vascularization for proper healing. Evidence indicates that osteogenesis and vessel growth are coupled in bone fractures. Although the synergistic role of endothelial cells has been recognized, vascularizing large bone grafts remains a challenge and has apprehended the clinical translation of engineered bone constructs. Here, we describe a facile method to fabricate vascularized constructs using chitosan and gelatin-based microgels that promote osteogenesis of human mesenchymal stromal cells (MSC) while supporting endothelial sprouting and network formation. The microgels are enzymatically degradable and had a high hydration rate with a volume swelling ratio of ~ 493% and a polymer density of ~ 431 mg/cm3, which is comparable to that of native skeletal tissues. AFM indentation of the surface showed an average Young's modulus of 189 kPa, falling in a range that is conducive to both osteogenesis and vasculogenesis. The osteogenic microgel containing chitosan, gelatin, and hydroxyapatite, mimicking the bone matrix, supported robust attachment, proliferation, and differentiation of MSC. On the other hand, the vasculogenic microgels containing only gelatin, enriched endothelial phenotype and enabled vascular networks formation when embedded in 3D matrices. Combining the two types of microgels created a hybrid construct that sustained the functions of both osteogenic and vasculogenic microgels and enhanced one another. Using a murine model, we also show that the osteogenic microgels regenerate bone in a critical-sized defect with > 95% defect closure by week 12. These multifunctional microgels can be administered minimally invasively and can conformally fill large bone defects. This work lays the foundation to establish principles of designing multiphasic scaffolds with tissue-specific biophysical and biochemical properties for regenerating vascularized and interfacial tissues.

Injectable osteogenic microtissues containing mesenchymal stromal cells conformally fill and repair critical-size defects.

Repair of complex fractures with bone loss requires a potent, space-filling intervention to promote regeneration of bone. We present a biomaterials-based strategy combining mesenchymal stromal cells (MSC) with a chitosan-collagen matrix to form modular microtissues designed for delivery through a needle to conformally fill cavital defects. Implantation of microtissues into a calvarial defect in the mouse showed that osteogenically pre-differentiated MSC resulted in complete bridging of the cavity, while undifferentiated MSC produced mineralized tissue only in apposition to native bone. Decreasing the implant volume reduced bone regeneration, while increasing the MSC concentration also attenuated bone formation, suggesting that the cell-matrix ratio is important in achieving a robust response. Conformal filling of the defect with microtissues in a carrier gel resulted in complete healing. Taken together, these results show that modular microtissues can be used to augment the differentiated function of MSC and provide an extracellular environment that potentiates bone repair.

Transport Analysis of Engineered Liver Tissue Fabricated Using a Capsule-Based, Modular Approach.

The bioinspired, microscale tissue engineering approach has emerged in recent years as a promising alternative to preformed scaffolds. Using this approach, complex tissues and organs can be efficiently engineered from microscale modules to replicate the intricate architecture and physiology of vascularized organs and tissues. Previously, we demonstrated assembly of a prototype, engineered liver tissue, formed by the fusion of hepatocyte-containing capsules. Here, we analyzed the effects of various controllable system parameters with the aim of predicting the operating limits of our modular tissue in high cell density, perfused cultures. Both the capsule diameter and construct height were limited by mass transfer requirements, while the shear stress on the capsule wall and the pressure drop across the packed capsule bed were dictated by the capsule diameter and permissible flow rates of the system. Our analysis predicts that capsules with a 200 µm radius can efficiently maintain hepatocytes at cell densities comparable to liver tissue. Some model predictions were validated by packed bed perfusion cultures. Flow-induced bed compaction hysteresis was tested experimentally and found to have minimal effect on flow characteristics. The effectiveness factor (η) for the overall oxygen transfer within packed beds of capsule modules was estimated to be 0.72 for all conditions. Primary hepatocytes encapsulated in the capsules exhibited normal metabolism and formed spheroids during a 7-day culture. The model predictions can be useful to study mass transfer and shear stress in high-density perfusion cultures. Overall, analysis of a perfused, capsule-based, modular tissue demonstrated the feasibility of the technology as a platform for fabrication of highly metabolic solid organs.

Multimode ultrasound viscoelastography for three-dimensional interrogation of microscale mechanical properties in heterogeneous biomaterials.

Both static and time-dependent mechanical factors can have a profound impact on cell and tissue function, but it is challenging to measure the mechanical properties of soft materials at the scale which cells sense. Multimode ultrasound viscoelastography (MUVE) uses focused ultrasound pulses to both generate and image deformations within soft hydrogels non-invasively, at sub-millimeter resolution, and in 3D. The deformation and strain over time data are used to extract quantitative parameters that describe both the elastic and viscoelastic properties of the material. MUVE was used in creep mode to characterize the viscoelastic properties of 3D agarose, collagen, and fibrin hydrogels. Quantitative comparisons were made by extracting characteristic viscoelastic parameters using Burger's lumped parameter constitutive model. Spatial resolution of the MUVE technique was found to be approximately 200 µm, while detection sensitivity, defined as the capability to differentiate between materials based on mechanical property differences, was approximately 0.2 kPa using agarose hydrogels. MUVE was superior to nanoindentation and shear rheometry in generating consistent microscale measurements of viscoelastic behavior in soft materials. These results demonstrate that MUVE is a rapid, quantitative, and accurate method to measure the viscoelastic mechanical properties of soft 3D hydrogels at the microscale, and is a promising technique to study the development of native and engineered tissues over time.

Biofabrication of injectable fibrin microtissues for minimally-invasive therapies: application of surfactants.

Microtissues created from the protein fibrin and containing embedded cells can be used in modular tissue engineering approaches to create larger, hierarchical and complex tissue structures. In this paper we demonstrate an emulsification-based method for the production of such fibrin microtissues containing fibroblasts (FB) and endothelial cells (EC) and designed to promote tissue vascularization. Surfactants can be beneficial in the microtissue fabrication process to reduce aggregation and to facilitate recovery of microtissues from the emulsion, thereby increasing yield. The nonionic surfactants Pluronic L101® and Tween 20® both increased microtissue yield in a dose-dependent fashion. Cell viability of both human FB and human EC remained high after exposure to low surfactant concentrations but decreased with increasing surfactant concentration. L101 was markedly less cytotoxic than Tween, and therefore was the surfactant of choice in this application. The yield of cell-laden microtissues increased with increasing L101 concentration, though microtissues were slightly larger at low concentrations. The total metabolic activity of cells in retrieved microtissues was bimodal and was highest at an L101 concentration of 0.10% wt/vol. Network formation by EC in microtissues embedded in surrounding 3D fibrin hydrogels was also most extensive in microtissues made using an L101 concentration of 0.10% wt/vol. Minimally-invasive delivery of microtissue populations was demonstrated by injection through a standard 18 G needle, and the ability to form robust endothelial networks was maintained in injected microtissue populations. Taken together, these data demonstrate a facile emulsification-based method to create modular, cell-laden hydrogel microtissues that can be delivered by injection to promote tissue regeneration. Appropriate selection of the type and concentration of surfactant used in the process can be used to maximize viability and specialized function of the embedded cells. Such biomaterial-based microtissues may have broad applicability in cell-based therapies and tissue engineering.

Harnessing macrophage-mediated degradation of gelatin microspheres for spatiotemporal control of BMP2 release.

Biomaterials-based approaches to harnessing the immune and inflammatory responses to potentiate wound healing hold important promise. Bone fracture healing is characterized by an acute inflammatory phase, followed by a transition to a regenerative and repair phase. In this study, we developed genipin-crosslinked gelatin microspheres designed to be preferentially degraded by inflammatory (M1) macrophages. Highly crosslinked (>90%) microspheres allowed efficient incorporation of bioactive bone morphogenetic protein 2 (BMP2), a potent stimulator of osteogenesis in progenitor cells, via electrostatic interactions. Release of BMP2 was directly correlated with degradation of the gelatin matrix. Exposure of microspheres to polarized murine macrophages showed that degradation was significantly higher in the presence of M1 macrophages, relative to alternatively activated (M2) macrophages and unpolarized controls. Microsphere degradation in the presence of non-inflammatory cells resulted in very low degradation rates. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) by macrophages were consistent with the observed phenotype-dependent degradation rates. Indirect co-culture of BMP2-loaded microspheres and macrophages with isolated adipose-derived mesenchymal stem cells (MSC) showed that M1 macrophages produced the strongest osteogenic response, comparable to direct supplementation of the culture medium with BMP2. Controlled release systems that are synchronized with the inflammatory response have the potential to provide better spatiotemporal control of growth factor delivery and therefore may improve the outcomes of recalcitrant wounds.