Models Of Bone Repair Research Articles

Tuberous sclerosis complex 1 targeted depletion in macrophages promotes osteogenesis by modulating secretion of Oncostatin M in the inflammatory stage of bone healing

In bone healing, earlier bone formation benefits bone repair. The first process of repair following bone injury involves the interaction between macrophage polarization and osteogenic activation of osteoblast linage cells, but the radical difference between the contributions of classically-activated M1 macrophages and alternatively-activated M2 macrophages to osteogenesis remains obscure. To test our hypothesis that M1 macrophages promote bone healing, we generated transgenic mice with myeloid lineage-specific TSC1 deletion (TSC1KO) to investigate the functional roles of M1 macrophages in the process of bone defect healing. We demonstrated that constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) enhances M1 macrophage polarization during bone healing. By creating tibial bone defect as a model of bone repair in TSC1KO mice and their littermates, we surprisingly found osteogenic responses in the defective bone region of TSC1KO mice, where repair occurred by intramembranous ossification (IO) in the mice was promoted due to the enhanced M1-polarized macrophage polarization. We propose that Oncostatin M (OSM) secreted by M1-polarized macrophages but not M2 macrophages likely functions as a paracrine factor in this promoted repair process, as verified by the induction of osteoblastic differentiation and matrix mineralization. Interestingly, the expression level of the OSM receptor (OSMR) was continually upregulated in osteoblast linage cells with M1 medium. Additionally, OSMR activated the signaling transduction system of JAK/STAT/RUNX2 in MSCs, which in turn stimulates the recruitment of osteoblast lineage cells and activates IO. These results indicate that TSC1 targeted depletion in macrophages promotes bone healing by inducing secretion of OSM. This study highlights that regulation of M1 macrophage polarization is a novel basis for the improvement of bone regeneration and that regulation of macrophage polarization can be a potential therapeutic strategy to treat defects in the repair phase of bone healing.

A Review of Advanced Biomaterials and Cells for the Production of Bone Organoid

Rapid advancements in traditional bone tissue engineering have led to innovation in bone repair models and the resolution of insurmountable clinical issues like graft scarcity. The pathophysiological process of treating bone disease, however, is a multidimensional and multimodal regenerative regulatory mechanism that includes numerous immune, inflammatory, or metabolic responses related to the graft or the organism itself. Based on a 3D in vitro cell culture system that is remarkably identical to the body's bone tissue, the bone organoid is a biomimicking bone organ environment. It can accurately mimic the actual repair and regeneration condition in vivo because it shares the same physiological function, structure, morphology, and metabolic process as endogenous bone tissue. As a disruptive regenerative medicine technology, it has wide application prospects in the fields of organ development, gene editing, disease modeling, and precision therapy. Herein, the development process and physiological basis of different cell‐based bone organoids are reviewed, the current status of the application of different materials, cells, and construction methods for building bone organoids is described, and the prospects and challenges for the development of bone organoids in future medical fields is discussed.

Evaluation of Bone Response to a Nano HA Implant Surface on Sinus Lifting Procedures: Study in Rabbits.

The aim of this study was to evaluate the bone response to two different implant surfaces on sinus lift procedures in rabbits. Bilateral sinus lifting with inorganic bovine bone associated with collagen membrane and immediate implantation were performed in 16 rabbits. Custom mini-implants were randomly installed in the prepared sites: one side received a double acid-etched (DAE) surface and the other a nano-hydroxyapatite (NHA) surface. The animals were euthanized 30 and 60 days after surgery, and biopsies were collected for microtomographic and histomorphometric analysis. After 30 days, no intra- and inter-group statistical differences were observed in microtomographic analysis, while at 60 days, bone analysis showed statistically significant differences between groups (p < 0.05) for all the evaluated parameters. Histomorphometric analysis showed, after 30 days, mean % of Bone-to-Implant Contact (BIC) for DAE and NHA of 31.70 ± 10.42% vs. 40.60 ± 10.22% (p > 0.05), respectively; for % of Bone Area Fraction Occupancy (BAFO), mean values were 45.43 ± 3.597% for DAE and 57.04 ± 5.537% for NHA (p < 0.05). After 60 days, mean %BIC and %BAFO for DAE and NHA implants were statistically significant (p < 0.05). The NHA surface showed superior biological features compared to the DAE treatment, promoting higher bone formation around the implants in an experimental model of bone repair in a grafted area.

Enhanced anti-microbial activity and osseointegration of Ta/Cu co-implanted polyetheretherketone

Polyetheretherketone (PEEK) has been widely applied for orthopedic and oral implants due to its excellent mechanical properties, biocompatibility, and radiolucency. However, its bioinert and the lack of anti-microbial activity limit its application. We modified the PEEK surface with Ta/Cu co-implantation using plasma immersion ion-implantation technology. After implantation of Ta/Cu ions, the morphology and roughness of the PEEK surface were not significantly changed at micron level. We estimated the cytocompatibility, anti-microbial ability, and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) of the modified surfaces in vitro. Compared to the untreated surfaces, the Ta ion-treated surface showed improved adhesion, proliferation, ALP activity, ECM mineralization, and osteogenic gene expression of BMSCs. Further, the Cu ion-treated surface showed reduced initial adhesion and proliferation of Escherichia coli and Staphylococcus aureus in vitro and proliferation of Staphylococcus aureus in the mouse subcutaneous implant-associated infection model. According to a rat bone repair model, all Ta ion-implanted groups demonstrated improved new bone formation. In summary, Ta/Cu ion co-impanation improved anti-microbial activity and promoted osseointegration of the PEEK surface.

Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell-cell interactions underlying the bone repair process.

IntroductionActivation of skeletal progenitors upon tissue injury and the subsequent cell fate specification are tightly coordinated in the bone repair process. Although known osteoimmunological signaling networks play important roles in the microenvironment of the bone defect sites, the molecular mechanism underlying the bone repair process has not been fully understood.MethodsTo better understand the behavior of the skeletal progenitors and the heterogeneity of the cells during bone repair at the microenvironmental level, we performed a combinatorial analysis consisting of lineage tracing for skeletal progenitors using the Sox9-CreERT2;R26RtdTomato mouse line followed by single-cell RNA sequencing (scRNA-seq) analysis using a mouse model of calvarial bone repair. To identify a therapeutic target for bone regeneration, further computational analysis was performed focusing on the identification of the cell–cell interactions, followed by pharmacological assessments with a critical-size calvarial bone defect mouse model.ResultsLineage tracing analysis showed that skeletal progenitors marked by Sox9 were activated upon bone injury and contributed to bone repair by differentiating into osteoblasts. The scRNA-seq analysis characterized heterogeneous cell populations at the bone defect sites; the computational analysis predicted a bifurcated lineage from skeletal progenitors toward osteogenic and adipogenic lineages. Chemokine C–C motif ligand 9 (Ccl9) was identified as a signaling molecule that regulates bone regeneration in the mouse model, possibly through the regulation of adipogenic differentiation at the bone defect site.ConclusionMultipotential skeletal progenitors and the direction of the cell differentiation were characterized at single cell resolution in a mouse bone repair model. The Ccl9 signaling pathway may be a key factor directing osteogenesis from the progenitors in the model and may be a therapeutic target for bone regeneration.

Inflammatory osteoclasts‐derived exosomes promote bone formation by selectively transferring lncRNA LIOCE into osteoblasts to interact with and stabilize Osterix

Bone loss is a hallmark of inflammatory bone diseases caused by aberrantly activated osteoclasts (OCLs). Studies have shown that OCLs exhibit various phenotypes and functions due to variations in the source(s) of precursor cells, cytokine expressions, and microenvironment-dependent factors. During these conditions, inflammatory osteoclasts (iOCLs) lose their immune-suppressive effect relative to OCLs under physiological conditions. This induces TNF α-producing CD4+ Tcells in an antigen-dependent manner and finally leads to cascade amplification of iOCLs. OCL-derived exosomes have been reported to regulate OCL formation and inhibit the osteoblast activity. However, the specific function and mechanism of iOCL-derived exosomes on osteoblast have not been studied yet. In the present study, we compare the osteoblast promoting activities of iOCL-derived exosomes and OCL-derived exosomes. We found that iOCLs exosomes specifically target osteoblasts through ephrinA2/EphA2. Mechanistically, the lncRNA LIOCE is enriched in iOCL exosomes and promotes the osteoblast activity after being incorporated into osteoblasts. Furthermore, our results revealed that exosomal lncRNA LIOCE stabilizes osteogenic transcription factor Osterix by interacting and reducing the ubiquitination level of Osterix. This study demonstrated that the bone loss is alleviated in the inflammatory osteolysis mice model after injection of iOCL exosomes encapsulating lncRNA LIOCE. The role of exosomes encapsulating lncRNA LIOCE in promoting bone formation was well established in the rat bone repair model. Our results indicate that iOCL-derived exosomal lncRNA LIOCE promotes bone formation by upregulating Osx expression, and thus, the exosomes encapsulating lncRNA LIOCE may be an effective strategy to increase bone formation in osteoporosis and other bone metabolic disorders.

Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage.

Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft®) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft®. This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product.

Drill Hole Models to Investigate Bone Repair.

Our understanding of the mechanisms underlying fracture healing is rapidly developing and is contributing to new therapeutic strategies to enhance repair. To gain new insights, animal models must also evolve. From initially imprecise, uncontrolled bone defects we now have precise injury models that still capture all of the stages and phases of bone repair yet do so in a highly reproducible manner. The simple mono-cortical defect model allows assessment of bone repair through a cartilage intermediate, e.g., endochondral ossification, as well as direct bone repair, e.g., intramembranous healing. Cellular contributions of the periosteum can be distinguished from contributions originating in the bone marrow. In this chapter, we focus on the advantages of this bone repair model, as well as its limitations.

Long non-coding RNA HCAR promotes endochondral bone repair by upregulating VEGF and MMP13 in hypertrophic chondrocyte through sponging miR-15b-5p

Endochondral bone formation is an important route for bone repair. Although emerging evidence has revealed the functions of long non-coding RNAs (lncRNAs) in bone and cartilage development, the effect of lncRNAs in endochondral bone repair is still largely unknown. Here, we identified a lncRNA, named Hypertrophic Chondrocyte Angiogenesis-related lncRNA (HCAR), and proved it to promote the endochondral bone repair by upregulating the expression of matrix metallopeptidase 13 (Mmp13) and vascular endothelial growth factor α (Vegfa) in hypertrophic chondrocytes. Lnc-HCAR knockdown in hypertrophic chondrocytes restrained the cartilage matrix remodeling and decrease the CD31hiEmcnhi vessels number in a bone repair model. Mechanistically, we proved that lnc-HCAR was mainly enriched in the cytoplasm using fluorescence in situ hybridization (FISH) assay, and it acted as a molecular sponge for miR-15b-5p. Further, in hypertrophic chondrocytes, lnc-HCAR competitively bound to miR-15b-5p to increase Vegfa and Mmp13 expression. Our results proved that lncRNA is deeply involved in endochondral bone repair, which will provide a new theoretical basis for future strategies for promoting fracture healing.

A segmental defect adaptation of the mouse closed femur fracture model for the analysis of severely impaired bone healing.

ObjectiveTo better characterize nonunion endochondral bone healing and evaluate novel therapeutic approaches for critical size defect healing in clinically challenging bone repair, a segmental defect model of bone injury was adapted from the three‐point bending closed fracture technique in the murine femur.MethodsThe mouse femur was surgically stabilized with an intramedullary threaded rod with plastic spacers and the defect adjusted to different sizes. Healing of the different defects was analyzed by radiology and histology to 8 weeks postsurgery. To determine whether this model was effective for evaluating the benefits of molecular therapy, BMP‐2 was applied to the defect and healing then examined.ResultsIntramedullary spacers were effective in maintaining the defect. Callus bone formation was initiated but was arrested at defect sizes of 2.5 mm and above, with no more progress in callus bone development evident to 8 weeks healing. Cartilage development in a critical size defect attenuated very early in healing without bone development, in contrast to the closed femur fracture healing, where callus cartilage was replaced by bone. BMP‐2 therapy promoted osteogenesis of the resident cells of the defect, but there was no further callus development to indicate that healing to pre‐surgery bone structure was successful.ConclusionsThis segmental defect adaptation of the closed femur fracture model of murine bone repair severely impairs callus development and bone healing, reflecting a challenging bone injury. It is adjustable and can be compared to the closed fracture model to ascertain healing deficiencies and the efficacy of therapeutic approaches.

Simultaneous nano- and microscale structural control of injectable hydrogels via the assembly of nanofibrous protein microparticles for tissue regeneration

Injectable hydrogels are advantageous as tissue regeneration scaffolds, as they can be delivered through a minimally invasive injection and seamlessly integrate with the target tissues. However, an important shortcoming of current injectable hydrogels is the lack of simultaneous control over their micro- and nanoscale structures. In this article, the authors report a strategy for developing injectable hydrogels that integrate a fibrous nanostructure and porous microstructure. The hydrogels are prepared by using novel nanofibrous microparticles as the building blocks. The protein based nanofibrous microparticles, fabricated by a spray freezing technology, can be injected through a syringe-needle system. A cell-compatible photocuring process can be deployed to connect the microparticles and form a mechanically robust hydrogel scaffold. The inter-particle voids combined to form the interconnected micropores and the diameter of the nanofibers (100–300 nm) closely mimics that of the native extracellular matrix. Compared to the non-porous hydrogels and non-fibrous hydrogels, the microparticle annealed nanofibrous (MANF) hydrogels potently enhance the osteogenic-marker expression (ALP, Runx2, OCT and BSP) and mineralization of human mesenchymal stem cells in vitro. MANF hydrogels also facilitate cell infiltration and enhance neovasculization in a subcutaneous implantation model in vivo. The capacity of MANF hydrogels to promote bone regeneration is investigated in a calvarial bone repair model. MANF hydrogels demonstrate significant higher bone regeneration after 8 weeks, indicating the significant role of microporosity and nanofibrous architecture in bone regeneration.

Specific, Sensitive, and Stable Reporting of Human Mesenchymal Stromal Cell Chondrogenesis

The promoter characterized in this study has been made accessible as a resource for the skeletal tissue engineering and regenerative medicine community. When combined with suitable reporter vectors, the resulting tools can be used for noninvasive and/or high-throughput screening of test conditions for stimulating chondrogenesis by candidate stem/progenitor cells. As demonstrated in this study, they can also be used with small animal imaging platforms to monitor the chondrogenic activity of implanted progenitors within orthotopic models of bone and cartilage repair.

Mechanical Loading Promotes the Expansion of Primitive Osteoprogenitors and Organizes Matrix and Vascular Morphology in Long Bone Defects.

Elucidating the effects of mechanical stimulation on bone repair is crucial for optimization of the healing process. Specifically, the regulatory role that mechanical loading exerts on the osteogenic stem cell pool and vascular morphology during healing is incompletely understood. Because dynamic loading has been shown to enhance osteogenesis and repair, we hypothesized that loading induces the expansion of the osteoprogenitor cell population within a healing bone defect, leading to an increased presence of osteogenic cells. We further hypothesized that loading during the repair process regulates vascular and collagen matrix morphology and spatial interactions between vessels and osteogenic cells. To address these hypotheses, we used a mechanobiological bone repair model, which produces a consistent and reproducible intramembranous repair response confined in time and space. Bilateral tibial defects were created in adult C57BL/6 mice, which were subjected to axial compressive dynamic loading either during the early cellular invasion phase on postsurgical days (PSDs) 2 to 5 or during the matrix deposition phase on PSD 5 to 8. Confocal and two-photon microscopy was used to generate high-resolution three-dimensional (3D) renderings of longitudinal thick sections of the defect on PSD 10. Endomucin (EMCN)-positive vessels, Paired related homeobox 1 (Prrx1+) stem cell antigen-1 positive (Sca-1+) primitive osteoprogenitors (OPCs), and osterix positive (Osx+) preosteoblasts were visualized and quantified using deep tissue immunohistochemistry. New bone matrix was visualized with second harmonic generation autofluorescence of collagen fibers. We found that mechanical loading during the matrix deposition phase (PSD 5 to 8) increased vessel volume and number, and aligned vessels and collagen fibers to the load-bearing direction of bone. Furthermore, loading led to a significant increase in the proliferation and number of Prrx1+ Sca-1+ primitive OPCs, but not Osx+ preosteoblasts within the defect. Together, these data illustrate the adaptation of both collagen matrix and vascular morphology to better withstand mechanical load during bone repair, and that the mechanoresponsive cell population consists of the primitive osteogenic progenitors. © 2019 American Society for Bone and Mineral Research.

Biological Compatibility Profile on Biomaterials for Bone Regeneration.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of β-tricalcium phosphate (β-TCP) disks. We also tested a β-TCP/Collagen (β-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

Lineage-Specific Wnt Reporter Elucidates Mesenchymal Wnt Signaling during Bone Repair

β-Catenin-dependent Wnt signaling controls numerous aspects of skeletal development and postnatal bone repair. Currently available transgenic Wnt reporter mice allow for visualization of global canonical Wnt signaling activity within skeletal tissues, without delineation of cell type. This is particularly important in a bone repair context, in which the inflammatory phase can obscure the visualization of mesenchymal cell types of interest. To tackle the issue of tissue-specific Wnt signaling, we have generated and characterized a transgenic mouse strain [termed paired related homeobox 1 (Prx1)-Wnt-green fluorescent protein (GFP), by crossing a previously validated Prx1-Cre strain with a nuclear fluorescent reporter driven by T-cell factor/lymphoid enhancer factor activity (Rosa26-Tcf/Lef-LSL-H2B-GFP)]. Prx1-Wnt-GFP animals were subject to three models of long bone and membranous bone repair (displaced forelimb fracture, tibial cortical defect, and frontal bone defect). Results showed that, irrespective of bone type, locoregional mesenchymal cell activation of Wnt signaling occurs in a defined temporospatial pattern among Prx1-Wnt-GFP mice. In summary, Prx1-Wnt-GFP reporter animals allow for improved visualization, spatial discrimination, and facile quantification of Wnt-activated mesenchymal cells within models of adult bone repair.

Intramembranous ossification and endochondral ossification are impaired differently between glucocorticoid-induced osteoporosis and estrogen deficiency-induced osteoporosis

A fracture is the most dangerous complication of osteoporosis in patients because the associated disability and mortality rates are high. Osteoporosis impairs fracture healing and prognosis, but how intramembranous ossification (IO) or endochondral ossification (EO) during fracture healing are affected and whether these two kinds of ossification are different between glucocorticoid-induced osteoporosis (GIOP) and estrogen deficiency-induced osteoporosis (EDOP) are poorly understood. In this study, we established two bone repair models that exhibited repair via IO or EO and compared the pathological progress of each under GIOP and EDOP. In the cortical drill-hole model, which is repaired through IO, osteogenic differentiation was more seriously impaired in EDOP at the early stage than in GIOP. In the periosteum scratch model, in which EO is replicated, chondrocyte hypertrophy progression was delayed in both GIOP and EDOP. The in vitro results were consistent with the in vivo results. Our study is the first to establish bone repair models in which IO and EO occur separately, and the results strongly describe the differences in bone repair between GIOP and EDOP.

Ecto-5'-nucleotidase (CD73) regulates bone formation and remodeling during intramembranous bone repair in aging mice.

Ecto-5'-nucleotidase (CD73) generates adenosine, an osteoblast activator and key regulator of skeletal growth. It is unknown, however, if CD73 regulates osteogenic differentiation during fracture healing in adulthood, and in particular how CD73 activity regulates intramembranous bone repair in the elderly. Monocortical tibial defects were created in 46-52-week-old wild type (WT) and CD73 knock-out mice (CD73-/-) mice. Injury repair was analyzed at post-operative days 5, 7, 14 and 21 by micro-computed tomography (micro-CT), histomorphometry, proliferating cell nuclear antigen (PCNA) immunostaining, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) histochemistry. Middle-aged CD73 knock-out mice exhibited delayed bone regeneration and significantly reduced bone matrix deposition detected by histomorphometry and micro-CT. Cell proliferation, ALP activity and osteoclast number were reduced in the CD73-/- mice, suggesting a combined defect in bone formation and resorption due the absence of CD73 activity in this model of intramembranous bone repair. Results from this study demonstrate that osteoblast activation through CD73 activity is essential during bone repair in aging mice, and it may present a drugable target for future biomimetic therapeutic approaches that aim at enhancing bone formation in the elderly patients.

NELL-1 induces Sca-1+ mesenchymal progenitor cell expansion in models of bone maintenance and repair.

NELL-1 is a secreted, osteogenic protein first discovered to control ossification of the cranial skeleton. Recently, NELL-1 has been implicated in bone maintenance. However, the cellular determinants of NELL-1's bone-forming effects are still unknown. Here, recombinant human NELL-1 (rhNELL-1) implantation was examined in a clinically relevant nonhuman primate lumbar spinal fusion model. Prolonged rhNELL-1 protein release was achieved using an apatite-coated β-tricalcium phosphate carrier, resulting in a local influx of stem cell antigen-1-positive (Sca-1+) mesenchymal progenitor cells (MPCs), and complete osseous fusion across all samples (100% spinal fusion rate). Murine studies revealed that Nell-1 haploinsufficiency results in marked reductions in the numbers of Sca-1+CD45-CD31- bone marrow MPCs associated with low bone mass. Conversely, rhNELL-1 systemic administration in mice showed a marked anabolic effect accompanied by increased numbers of Sca-1+CD45-CD31- bone marrow MPCs. Mechanistically, rhNELL-1 induces Sca-1 transcription among MPCs, in a process requiring intact Wnt/β-catenin signaling. In summary, NELL-1 effectively induces bone formation across small and large animal models either via local implantation or intravenous delivery. NELL-1 induces an expansion of a bone marrow subset of MPCs with Sca-1 expression. These findings provide compelling justification for the clinical translation of a NELL-1-based therapy for local or systemic bone formation.

The Selective Serotonin Reuptake Inhibitor Fluoxetine Directly Inhibits Osteoblast Differentiation and Mineralization During Fracture Healing in Mice.

Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft-callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard-callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days postinjury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and μCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well-established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing. © 2017 American Society for Bone and Mineral Research.

Defective Bone Repair in C57Bl6 Mice With Acute Systemic Inflammation.

Bone repair is initiated with a local inflammatory response to injury. The presence of systemic inflammation impairs bone healing and often leads to malunion, although the underlying mechanisms remain poorly defined. Our research objective was to use a mouse model of cortical bone repair to determine the effect of systemic inflammation on cells in the bone healing microenvironment. QUESTION/PURPOSES: (1) Does systemic inflammation, induced by lipopolysaccharide (LPS) administration affect the quantity and quality of regenerating bone in primary bone healing? (2) Does systemic inflammation alter vascularization and the number or activity of inflammatory cells, osteoblasts, and osteoclasts in the bone healing microenvironment? Cortical defects were drilled in the femoral diaphysis of female and male C57BL/6 mice aged 5 to 9 months that were treated with daily systemic injections of LPS or physiologic saline as control for 7 days. Mice were euthanized at 1 week (Control, n = 7; LPS, n = 8), 2 weeks (Control, n = 7; LPS, n = 8), and 6 weeks (Control, n = 9; LPS, n = 8) after surgery. The quantity (bone volume per tissue volume [BV/TV]) and microarchitecture (trabecular separation and thickness, porosity) of bone in the defect were quantified with time using microCT. The presence or activity of vascular endothelial cells (CD34), macrophages (F4/80), osteoblasts (alkaline phosphatase [ALP]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP]) were evaluated using histochemical analyses. Only one of eight defects was bridged completely 6 weeks after surgery in LPS-injected mouse bones compared with seven of nine defects in the control mouse bones (odds ratio [OR], 0.04; 95% CI, 0.003-0.560; p = 0.007). The decrease in cortical bone in LPS-treated mice was reflected in reduced BV/TV (21% ± 4% vs 39% ± 10%; p < 0.01), increased trabecular separation (240 ± 36 μm vs 171 ± 29 μm; p < 0.01), decreased trabecular thickness (81 ± 18 μm vs 110 ± 22 μm; p = 0.02), and porosity (79% ± 4% vs 60% ± 10%; p < 0.01) at 6 weeks postoperative. Defective healing was accompanied by decreased CD34 (1.1 ± 0.6 vs 3.4 ± 0.9; p < 0.01), ALP (1.9 ± 0.9 vs 6.1 ± 3.2; p = 0.03), and TRAP (3.3 ± 4.7 vs 7.2 ± 4.0; p = 0.01) activity, and increased F4/80 (13 ± 2.6 vs 6.8 ± 1.7; p < 0.01) activity at 2 weeks postoperative. The results indicate that LPS-induced systemic inflammation reduced the amount and impaired the quality of bone regenerated in mouse femurs. The effects were associated with impaired revascularization, decreased bone turnover by osteoblasts and osteoclasts, and by increased catabolic activity by macrophages. Results from this preclinical study support clinical observations of impaired primary bone healing in patients with systemic inflammation. Based on our data, local administration of VEGF in the callus to stimulate revascularization, or transplantation of stem cells to enhance bone turnover represent potentially feasible approaches to improve outcomes in clinical practice.