Early Gßγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects.

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gßγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gßγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gßγ-GRK2 inhibition at the time of DMM by administering the Gßγ inhibitor "gallein" and the GRK2 inhibitor "paroxetine" daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gßγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gßγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gßγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gßγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.

Cardiac Fibrosis: The Fibroblast Awakens.

Myocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Cardiac fibroblasts comprise an essential cell type in the heart that is responsible for the homeostasis of the extracellular matrix; however, upon injury, these cells transform to a myofibroblast phenotype and contribute to cardiac fibrosis. This remodeling involves pathological changes that include chamber dilation, cardiomyocyte hypertrophy and apoptosis, and ultimately leads to the progression to heart failure. Despite the critical importance of fibrosis in cardiovascular disease, our limited understanding of the cardiac fibroblast impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression. This review summarizes current knowledge regarding the origins and roles of fibroblasts, mediators and signaling pathways known to influence fibroblast function after myocardial injury, as well as novel therapeutic strategies under investigation to attenuate cardiac fibrosis.

G Protein-Coupled Receptor-G-Protein ßγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein ßγ-subunit (Gßγ), GPCR-kinase 2, and ß-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gßγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gßγ signaling and ET system expression. Notably, systemic pharmacologic Gßγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gßγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gßγ in AKI and suggest GPCR-Gßγ inhibition as a novel therapeutic approach for treating CRS2 and AKI.

Extraction of high-quality RNA from human articular cartilage.

Extracting high-quality RNA from articular cartilage is challenging due to low cellularity and high proteoglycan content. This problem hinders efficient application of RNA sequencing (RNA-seq) analysis in studying cartilage homeostasis. Here we developed a method that purifies high-quality RNA directly from cartilage. Our method optimized the collection and homogenization steps so as to minimize RNA degradation, and modified the conventional TRIzol protocol to enhance RNA purity. Cartilage RNA purified using our method has appropriate quality for RNA-seq experiments including an RNA integrity number of ∼8. Our method also proved efficient in extracting high-quality RNA from subchondral bone.

Glomerular expression of connexin 40 and connexin 43 in rat experimental glomerulonephritis.

BACKGROUND: Gap junctional intercellular communication is thought to play an important role in the maintenance of cell differentiation and homeostasis. Gap junctions connect glomerular mesangial cells to each other. In this study, we examined the glomerular expression of connexins (Cxs) 40 and 43 at both the protein and transcript levels in anti-Thy1.1 glomerulonephritis (GN). METHODS: Anti-Thy1.1 GN was induced by intravenous injection of anti-Thy1.1 monoclonal antibody 1-22-3. Cx protein expression was examined by immunofluorescence, immunoelectron microscopy, and Western blotting. Changes in mRNA levels were detected by real-time reverse transcriptase-polymerase chain reaction. RESULTS: Cx40 was detected in mesangial cells in normal rat glomeruli; its expression was reduced on days 3 and 7 and recovered to normal on day 14 following GN induction. Cx43 was detected in mesangial cells and podocytes in normal rat glomeruli, and its expression did not change during the disease course of GN. Expression of Cx40 and Cx43 was also detected in extraglomerular mesangial cells; this expression did not change during the disease course. Opposing patterns of expression between Cx40 and smooth muscle actin (SMA) were observed with double-immunofluorescence labeling. SMA is a differentiation marker of mesangial cells; it is often expressed during proliferation but not under physiological conditions. CONCLUSION: These results suggest that Cx40 expression in mesangial cells is related to mesangial cell regeneration. Thus, Cx expression regulation could be a therapeutic target for glomerular diseases.

Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity.

Bone fractures, the most common musculoskeletal injuries, heal through three main phases: inflammatory, repair, and remodeling. Around 10% of fracture patients suffer from impaired healing that requires surgical intervention, a huge burden on the healthcare system. The rate of impaired healing increases with metabolic diseases such as obesity-associated hyperglycemia/type 2 diabetes (T2D), an increasing concern given the growing incidence of obesity/T2D. Immune cells play pivotal roles in fracture healing, and obesity/T2D is associated with defective immune-cell functions. However, there is a gap in knowledge regarding the stoichiometry of immune cells that populate the callus and how that population changes during different phases of healing. Here, we used complementary global and single-cell techniques to characterize the repertoire of immune cells in the fracture callus and to identify populations specifically enriched in the fracture callus relative to the unfractured bone or bone marrow. Our analyses identified two clear waves of immune-cell infiltration into the callus: the first wave occurs during the early inflammatory phase of fracture healing, while the second takes place during the late repair/early remodeling phase, which is consistent with previous publications. Comprehensive analysis of each wave revealed that innate immune cells were activated during the early inflammatory phase, but in later phases they returned to homeostatic numbers and activation levels. Of the innate immune cells, distinct subsets of activated dendritic cells were particularly enriched in the inflammatory healing hematoma. In contrast to innate cells, lymphocytes, including B and T cells, were enriched and activated in the callus primarily during the late repair phase. The Diet-Induced Obesity (DIO) mouse, an established model of obesity-associated hyperglycemia and insulin resistance, suffers from multiple healing defects. Our data demonstrate that DIO mice exhibit dysregulated innate immune responses during the inflammatory phase, and defects in all lymphocyte compartments during the late repair phase. Taken together, our data characterize, for the first time, immune populations that are enriched/activated in the callus during two distinct phases of fracture healing and identify defects in the healing-associated immune response in DIO mice, which will facilitate future development of immunomodulatory therapeutics for impaired fracture healing.

Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing.

Maturation of the 3' end of almost all eukaryotic messenger RNAs (mRNAs) requires cleavage and polyadenylation. Most mammalian mRNAs are polyadenylated at different sites within the last exon, generating alternative polyadenylation (APA) isoforms that have the same coding region but distinct 3' untranslated regions (UTRs). The 3'UTR contains motifs that regulate mRNA metabolism; thus, changing the 3'UTR length via APA can significantly affect gene expression. Endochondral ossification is a central process in bone healing, but the impact of APA on gene expression during this process is unknown. Here, we report the widespread occurrence of APA, which impacts multiple pathways that are known to participate in bone healing. Importantly, the progression of endochondral ossification involves global 3'UTR shortening, which is coupled with an increased abundance of shortened transcripts relative to other transcripts; these results highlight the role of APA in promoting gene expression during endochondral bone formation. Our mechanistic studies of transcripts that undergo APA in the fracture callus revealed an intricate regulatory network in which APA enhances the expression of the collagen, type I, alpha 1 (Col1a1) and Col1a2 genes, which encode the 2 subunits of the abundantly expressed protein collagen 1. APA exerts this effect by shortening the 3'UTRs of the Col1a1 and Col1a2 mRNAs, thus removing the binding sites of miR-29a-3p, which would otherwise strongly promote the degradation of both transcripts. Taken together, our study is the first to characterize the crucial roles of APA in regulating the 3'UTR landscape and modulating gene expression during fracture healing.

Cannabidiol and Cannabigerol, Nonpsychotropic Cannabinoids, as Analgesics that Effectively Manage Bone Fracture Pain and Promote Healing in Mice.

Bone fractures are among the most prevalent musculoskeletal injuries, and pain management is an essential part of fracture treatment. Fractures heal through an early inflammatory phase, followed by repair and remodeling. Nonsteroidal anti-inflammatory drugs (NSAIDs) are not recommended for fracture pain control as they potently inhibit the inflammatory phase and, thus, impair the healing. Opioids do not provide a better alternative for several reasons, including abuse potential. Accordingly, there is an unmet clinical need for analgesics that effectively ameliorate postfracture pain without impeding the healing. Here, we investigated the analgesic efficacy of two nonpsychotropic cannabinoids, cannabidiol (CBD) and cannabigerol (CBG), in a mouse model for tibial fracture. Mice with fractured tibiae exhibited increased sensitivity to mechanical, cold, and hot stimuli. Both CBD and CBG normalized pain sensitivity to all tested stimuli, and their analgesic effects were comparable to those of the NSAIDs. Interestingly, CBD and CBG promoted bone healing via multiple mechanisms during the early and late phases. During the early inflammatory phase, both cannabinoids increased the abundance of periosteal bone progenitors in the healing hematoma and promoted the osteogenic commitment of these progenitors. During the later phases of healing, CBD and CBG accelerated the fibrocartilaginous callus mineralization and enhanced the viability and proliferation of bone and bone-marrow cells. These effects culminated in higher bone volume fraction, higher bone mineral density, and improved mechanical quality of the newly formed bone. Together, our data suggest CBD and CBG as therapeutic agents that can replace NSAIDs in managing postfracture pain as both cannabinoids exert potent analgesic effects and, at the same time, promote bone healing. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Therapeutic Effects of Non-Euphorigenic Cannabis Extracts in Osteoarthritis.

Introduction: Osteoarthritis (OA) is disabling and degenerative disease of the joints that is clinically characterized by pain and loss of function. With no disease-modifying treatment available, current therapies aim at pain management but are of limited efficacy. Cannabis products, specifically cannabinoids, are widely used to control pain and inflammation in many diseases with no scientific evidence demonstrating their efficacy in OA. Objective: We investigated the effects of non-euphorigenic cannabis extracts, CBD oil and cannabigerol oil (CBG oil), on pain and disease progression in OA mice. Methods and Results: Twelve-week-old male C57BL/6J mice received either sham or destabilization of the medial meniscus (DMM) surgery. DMM mice were treated with vehicle, CBD oil, or CBG oil. The gait of DMM mice was impaired as early as 2 weeks following surgery and continued deteriorating until week 8, which was restored by CBD oil and CBG oil treatments throughout the disease course. Mechanical allodynia developed in DMM mice, however, was not ameliorated by any of the treatments. On the other hand, both CBD oil and CBG oil ameliorated cold allodynia. In open field test, both oil treatments normalized changes in the locomotor activity of DMM mice. CBD oil and CBG oil treatments significantly reduced synovitis in DMM mice. Only CBG oil reduced cartilage degeneration, chondrocyte loss, and matrix metalloproteinase 13 expression, with a significant increase in the number of anabolic chondrocytes. Subchondral bone remodeling found in vehicle-treated DMM mice was not ameliorated by either CBD or CBG oil. Conclusions: Our results show evidence for the therapeutic efficacy of CBD oil and CBG oil, where both oils ameliorate pain and inflammation, and improve gait and locomotor activity in OA mice, representing clinical pain and function. Importantly, only CBG oil is chondroprotective, which may provide superior efficacy in future studies in OA patients.

miRNA induced 3D bioprinted-heterotypic osteochondral interface.

The engineering of osteochondral interfaces remains a challenge. MicroRNAs (miRs) have emerged as significant tools to regulate the differentiation and proliferation of osteogenic and chondrogenic formation in the human musculoskeletal system. Here, we describe a novel approach to osteochondral reconstruction based on the three-dimensional (3D) bioprinting of miR-transfected adipose-derived stem cell (ADSC) spheroids to produce a heterotypic interface that addresses the intrinsic limitations of the traditional approach to inducing zonal differentiation via the use of diffusible cytokines. We evaluated the delivery of miR-148b for osteogenic differentiation and the codelivery of miR-140 and miR-21 for the chondrogenic differentiation of ADSC spheroids. Our results demonstrated that miR-transfected ADSC spheroids exhibited upregulated expression of osteogenic and chondrogenic differentiation related gene and protein markers, and enhanced mineralization and cell proliferation compared to spheroids differentiated using a commercially-available differentiation medium. Upon confirmation of the osteogenic and chondrogenic potential of miR-transfected ADSC spheroids, using aspiration-assisted bioprinting, these spheroids were 3D bioprinted into a dual-layer heterotypic osteochondral interface with a stratified arrangement of distinct osteogenic and chondrogenic zones. The proposed approach holds great promise for the biofabrication of stratified tissues, not only for the osteochondral interfaces presented in this work, but also for other composite tissues and tissue interfaces, such as, but not limited to, the bone-tendon-muscle interface and craniofacial tissues.

Assessment of Bone Fracture Healing Using Micro-Computed Tomography.

Micro-computed tomography (µCT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Structural changes in the collagen network of joint tissues in late stages of murine OA.

Osteoarthritis (OA) is the most prevalent degenerative joint disease, resulting in joint pain, impaired movement, and structural changes. As the ability of joint tissue to resist stress is mainly imparted by fibrillar collagens in the extracellular matrix, changes in the composition and structure of collagen fibers contribute to the pathological remodeling observed in OA joints that includes cartilage degeneration, subchondral bone (SCB) sclerosis, and meniscal damage. Using the established OA model of destabilization of the medial meniscus (DMM) in C57BL/6J mice, we performed a comprehensive analysis of the content and structure of collagen fibers in the articular cartilage, subchondral bone, and menisci using complementary techniques, which included second harmonic generation microscopy and immunofluorescence staining. We found that regions exposed to increased mechanical stress in OA mice, typically closest to the site of injury, had increased collagen fiber thickness, dysregulated fiber formation, and tissue specific changes in collagen I and II (Col I and Col II) expression. In cartilage, OA was associated with decreased Col II expression in all regions, and increased Col I expression in the anterior and posterior regions. Col I fiber thickness was increased in all regions with disorganization in the center region. In the superficial SCB, all regions exhibited increased Col I expression and fiber thickness in OA mice; no changes were detected in the deeper regions of the subchondral bone except for increased Col I fiber thickness. In the menisci, OA led to increased Col I and Col II expression in the vascular and avascular regions of the anterior meniscus with increased Col I fiber thickness in these regions. Similar changes were observed only in the vascular region of the posterior meniscus. Our findings provide, for the first time, comprehensive insights into the microarchitectural changes of extracellular matrix in OA and serve as guidelines for studies investigating therapies that target collagenous changes as means to impede the progression of osteoarthritis.

Taking the heart failure battle inside the cell: small molecule targeting of Gßγ subunits.

Heart failure (HF) is devastating disease with poor prognosis. Elevated sympathetic nervous system activity and outflow, leading to pathologic attenuation and desensitization of ß-adrenergic receptors (ß-ARs) signaling and responsiveness, are salient characteristic of HF progression. These pathologic effects on ß-AR signaling and HF progression occur in part due to Gßγ-mediated signaling, including recruitment of receptor desensitizing kinases such as G-protein coupled receptor (GPCR) kinase 2 (GRK2) and phosphoinositide 3-kinase (PI3K), which subsequently phosphorylate agonist occupied GPCRs. Additionally, chronic GPCR signaling signals chronically dissociated Gßγ subunits to interact with multiple effector molecules that activate various signaling cascades involved in HF pathophysiology. Importantly, targeting Gßγ signaling with large peptide inhibitors has proven a promising therapeutic paradigm in the treatment of HF. We recently described an approach to identify small molecule Gßγ inhibitors that selectively block particular Gßγ functions by specifically targeting a Gßγ protein-protein interaction "hot spot." Here we describe their effects on Gßγ downstream signaling pathways, including their role in HF pathophysiology. We suggest a promising therapeutic role for small molecule inhibition of pathologic Gßγ signaling in the treatment of HF. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

A novel phenylpyridazinone, T-3999, reduces the progression of autoimmune myocarditis to dilated cardiomyopathy.

Regardless of the origin, injury to the heart can result in cardiomyocyte hypertrophy, fibrosis, and cell death. Myocarditis often progresses to dilated cardiomyopathy (DCM), a major cause of heart failure. In our study, we used a rat model of myosin-induced experimental autoimmune myocarditis (EAM), in which the heart transits from an acute phase (inflammatory myocarditis) to a chronic phase (remodeling and DCM). Our objective was to investigate whether T-3999, a novel phenylpyridazinone, can reduce this progression. Four weeks after myosin injection, T-3999 was administered daily to male Lewis rats in two doses (3 and 10 mg/kg, orally). Four weeks later, treatment was terminated; hemodynamic and echocardiographic measurements were performed; hearts were excised for histopathology and estimation of histamine, mRNA, and protein levels. Mortality rate was reduced by drug treatment. T-3999 reduced % fibrosis and tissue collagen III. Profibrotic markers-transforming growth factor-ß(1), tumor necrosis factor-α, and galectin-3--were attenuated by treatment. Mast cell density and degranulation, and tissue histamine concentration were also reduced. This indicates an anti-inflammatory effect of the drug in reducing fibrosis. Hypertrophy was reduced as reflected by reduced myocyte diameter and natriuretic peptide expression. T-3999 treatment increased the sarcoendoplasmic reticulum Ca(2+) ATPase 2 protein level and improved several cardiac function parameters. The reduction of the remodeling process and improvement in myocardial function suggest an effect of T-3999 in attenuating ventricular remodeling in post-myocarditis DCM.

Paroxetine-mediated GRK2 inhibition is a disease-modifying treatment for osteoarthritis.

Osteoarthritis (OA) is a debilitating joint disease characterized by progressive cartilage degeneration, with no available disease-modifying therapy. OA is driven by pathological chondrocyte hypertrophy (CH), the cellular regulators of which are unknown. We have recently reported the therapeutic efficacy of G protein-coupled receptor kinase 2 (GRK2) inhibition in other diseases by recovering protective G protein-coupled receptor (GPCR) signaling. However, the role of GPCR-GRK2 pathway in OA is unknown. Thus, in a surgical OA mouse model, we performed genetic GRK2 deletion in chondrocytes or pharmacological inhibition with the repurposed U.S. Food and Drug Administration (FDA)-approved antidepressant paroxetine. Both GRK2 deletion and inhibition prevented CH, abated OA progression, and promoted cartilage regeneration. Supporting experiments with cultured human OA cartilage confirmed the ability of paroxetine to mitigate CH and cartilage degradation. Our findings present elevated GRK2 signaling in chondrocytes as a driver of CH in OA and identify paroxetine as a disease-modifying drug for OA treatment.

Local delivery of angiotensin II receptor blockers into the kidney passively attenuates inflammatory reactions during the early phases of streptozotocin-induced diabetic nephropathy through inhibition of calpain activity.

BACKGROUND/AIMS: Inhibition of the renin-angiotensin-aldosterone system plays a pivotal role in the prevention and treatment of diabetic nephropathy. Angiotensin II receptor blockers (ARB) exert a renoprotective effect and attenuate the progression of diabetic nephropathy. However, the underlying cellular and molecular mechanisms in the kidney remain to be elucidated. The present study was undertaken to focus on the effect of local angiotensin II type 1 receptor blockade on the inflammatory reaction during the early stages of diabetic nephropathy. METHODS AND RESULTS: Local ARB treatment significantly reduced urinary protein excretion and serum blood urea nitrogen levels in streptozotocin-induced diabetic nephropathy. In addition, this treatment attenuated monocyte/macrophage infiltration into the glomeruli and the enhanced glomerular expression of endothelial nitric oxide synthase at both the mRNA and protein levels. Immunohistochemical study revealed activation of nuclear factor (NF)-kappaB, as shown by an increase in the expression of the p65 subunit of NF-kappaB and its translocation from the cytoplasm to the nucleus in both tubular epithelial and glomerular cells of the diabetic kidney. Local ARB treatment induced an apparent reduction in p65 nuclear localization and intensity of staining. To search for a common and fundamental candidate that influences endothelial cell function and vascular inflammation, we examined glomerular calpain activity in diabetic rats with or without ARB treatment. Glomerular expression of 145/150-kDa spectrin breakdown products, a specific product of calpain activation, was dramatically increased in diabetic animals while the protein expression reverted to a normal level after ARB treatment. CONCLUSION: Our findings provide a conceptual basis for the development of therapeutic strategies aiming at local inhibition of the renin-angiotensin system to prevent the progression of diabetic nephropathy.

Aspiration-assisted bioprinting of the osteochondral interface.

Osteochondral defects contain damage to both the articular cartilage and underlying subchon- dral bone, which remains a significant challenge in orthopedic surgery. Layered structure of bone, cartilage and the bone-cartilage interface must be taken into account in the case of biofabrication of the osteochondral (OC) interface. In this study, a dual layered OC interface was bioprinted using a newly developed aspiration-assisted bioprinting (AAB) technique, which has been the first time that scaffold-free bioprinting was applied to OC interface engineering. Tissue spheroids, made of human adipose-derived stem cells (ADSCs), were differentiated in three dimensions (3D) into chondrogenic and osteogenic spheroids, which were confirmed by immunostaining and histology qualitatively, and biochemistry assays and gene expression, quantitatively. Remarkably, the OC interface was bioprinted by accurate positioning of a layer of osteogenic spheroids onto a sacrificial alginate support followed by another layer of chondrogenic spheroids overlaid by the same support. Spheroids in individual zones fused and the maintenance of phenotypes in both zones confirmed the successful biofabrication of the histomorphologically-relevant OC interface. The biofabrication of OC tissue model without the use of polymeric scaffolds unveils great potential not only in regenerative medicine but also in drug testing and disease modeling for osteoarthritis.

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

Aberrant structure of fibrillar collagen and elevated levels of advanced glycation end products typify delayed fracture healing in the diet-induced obesity mouse model.

Impaired fracture healing in patients with obesity-associated type 2 diabetes (T2D) is a significant unmet clinical problem that affects millions of people worldwide. However, the underlying causes are poorly understood. Additionally, limited clinical information is available on how pre-diabetic hyperglycemia in obese individuals impacts bone healing. Here, we use the diet-induced obesity (DIO) mouse (C57BL/6J) model to study the impact of obesity-associated pre-diabetic hyperglycemia on bone healing and fibrillar collagen organization as healing proceeds from one phase to another. We show that DIO mice exhibit defective healing characterized by reduced bone mineral density, bone volume, and bone volume density. Differences in the healing pattern between lean and DIO mice occur early in the healing process as evidenced by faster resorption of the fibrocartilaginous callus in DIO mice. However, the major differences between lean and DIO mice occur during the later phases of endochondral ossification and bone remodeling. Comprehensive analyses of fibrillar collagen microstructure and expression pattern during these phases, using a set of complementary techniques that include histomorphometry, immunofluorescence staining, and second harmonic generation microscopy, demonstrate significant defects in DIO mice. Defects include strikingly sparse and disorganized collagen fibers, as well as pathological accumulation of unfolded collagen triple helices. We also demonstrate that DIO-associated changes in fibrillar collagen structure are attributable, at least in part, to the accumulation of advanced glycation end products, which increase the collagen-fiber crosslink density. These major changes impair fibrillar collagens functions, culminating in defective callus mineralization, remodeling, and strength. Our data extend the understanding of mechanisms by which obesity and its associated hyperglycemia impair fracture healing and underline defective fibrillar collagen microstructure as a novel and important contributor.