Tissue engineering approaches for dental pulp regeneration: The development of novel bioactive materials using pharmacological epigenetic inhibitors.

The drive for minimally invasive endodontic treatment strategies has shifted focus from technically complex and destructive root canal treatments towards more conservative vital pulp treatment. However, novel approaches to maintaining dental pulp vitality after disease or trauma will require the development of innovative, biologically-driven regenerative medicine strategies. For example, cell-homing and cell-based therapies have recently been developed in vitro and trialled in preclinical models to study dental pulp regeneration. These approaches utilise natural and synthetic scaffolds that can deliver a range of bioactive pharmacological epigenetic modulators (HDACis, DNMTis, and ncRNAs), which are cost-effective and easily applied to stimulate pulp tissue regrowth. Unfortunately, many biological factors hinder the clinical development of regenerative therapies, including a lack of blood supply and poor infection control in the necrotic root canal system. Additional challenges include a need for clinically relevant models and manufacturing challenges such as scalability, cost concerns, and regulatory issues. This review will describe the current state of bioactive-biomaterial/scaffold-based engineering strategies to stimulate dentine-pulp regeneration, explicitly focusing on epigenetic modulators and therapeutic pharmacological inhibition. It will highlight the components of dental pulp regenerative approaches, describe their current limitations, and offer suggestions for the effective translation of novel epigenetic-laden bioactive materials for innovative therapeutics.

Toxin for Treating Raynaud Conditions in Hands (The TORCH Study): A Systematic Review and Meta-analysis.

Background: Raynaud disease of the hands is a complex disorder resulting in inappropriate constriction and/or insufficient dilation in microcirculation. There is an emerging role for botulinum toxin type A (BTX-A) in the treatment armamentarium for refractory Raynaud disease. The aim of this systematic review was to critically evaluate the management of primary and secondary Raynaud disease treated with BTX-A intervention. Methods: We performed a Preferred Reporting Items for Systematic Reviews and Meta-Analyses-compliant systematic review of clinical studies assessing treatment of primary or secondary Raynaud disease with BTX-A by searching Ovid MEDLINE and Embase databases from inception to first August 2023. The review protocol was prospectively registered on the PROSPERO database (CRD42022312253). Results: Our search strategy identified 288 research articles, of which 18 studies [four randomized controlled trials (RCTs), two non-RCTs, five case series, and seven retrospective cohort studies] were eligible for analysis. Meta-analysis demonstrated that the probability of pain visual analog scale score improvement with BTX-A intervention was 81.95% [95% confidence interval (74.12-87.81) P = 0.19, heterogeneity I 2 = 26%] and probability of digital ulcer healing was 79.37% [95% confidence interval (62.45-89.9) P = 0.02, heterogeneity I 2 = 56%]. Conclusions: Delivery of BTX-A to digital vessels in the hand may be an effective management strategy for primary and secondary Raynaud disease. A definitive, appropriately-powered RCT with objective functional and patient-reported outcome measures is required to accurately assess and quantify the efficacy of BTX-A in Raynaud disease of the hands.

Reproducible Synthesis of Biocompatible Albumin Nanoparticles Designed for Intra-articular Administration of Celecoxib to Treat Osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis, with intra-articular (IA) delivery of therapeutics being the current best option to treat pain and inflammation. However, IA delivery is challenging due to the rapid clearance of therapeutics from the joint and the need for repeated injections. Thus, there is a need for long-acting delivery systems that increase the drug retention time in joints with the capacity to penetrate OA cartilage. As pharmaceutical utility also demands that this is achieved using biocompatible materials that provide colloidal stability, our aim was to develop a nanoparticle (NP) delivery system loaded with the COX-2 inhibitor celecoxib that can meet these criteria. We devised a reproducible and economical method to synthesize the colloidally stable albumin NPs loaded with celecoxib without the use of any of the following conditions: high temperatures at which albumin denaturation occurs, polymer coatings, oils, Class 1/2 solvents, and chemical protein cross-linkers. The spherical NP suspensions were biocompatible, monodisperse with average diameters of 72 nm (ideal for OA cartilage penetration), and they were stable over 6 months at 4 °C. Moreover, the NPs loaded celecoxib at higher levels than those required for the therapeutic response in arthritic joints. For these reasons, they are the first of their kind. Labeled NPs were internalized by primary human articular chondrocytes cultured from the knee joints of OA patients. The NPs reduced the concentration of inflammatory mediator prostaglandin E2 released by the primaries, an indication of retained bioactivity following NP synthesis. Similar results were observed in lipopolysaccharide-stimulated human THP-1 monocytes. The IA administration of these NPs is expected to avoid side-effects associated with oral administration of celecoxib and to maintain a high local concentration in the knee joint over a sustained period. They are now ready for evaluation by IA administration in animal models of OA.

Impact of Fluid Flow Shear Stress on Osteoblast Differentiation and Cross-Talk with Articular Chondrocytes.

Bone cells, in particular osteoblasts, are capable of communication with each other during bone growth and homeostasis. More recently it has become clear that they also communicate with other cell-types; including chondrocytes in articular cartilage. One way that this process is facilitated is by interstitial fluid movement within the pericellular and extracellular matrices. This stimulus is also an important mechanical signal in skeletal tissues, and is known to generate shear stresses at the micron-scale (known as fluid flow shear stresses (FFSS)). The primary aim of this study was to develop and characterize an in vitro bone-cartilage crosstalk system, to examine the effect of FFSS on these cell types. Specifically, we evaluated the response of osteoblasts and chondrocytes to FFSS and the effect of FFSS-induced soluble factors from the former, on the latter. This system will ultimately be used to help us understand the role of subchondral bone damage in articular cartilage degeneration. We also carried out a comparison of responses between cell lines and primary murine cells in this work. Our findings demonstrate that primary cells produce a more reliable and reproducible response to FFSS. Furthermore we found that at lower magnitudes , direct FFSS produces anabolic responses in both chondrocytes and osteoblasts, whereas higher levels produce more catabolic responses. Finally we show that exposure to osteoblast-derived factors in conditioned media experiments produced similarly catabolic changes in primary chondrocytes.

Mitochondria in Injury, Inflammation and Disease of Articular Skeletal Joints.

Inflammation is an important biological response to tissue damage caused by injury, with a crucial role in initiating and controlling the healing process. However, dysregulation of the process can also be a major contributor to tissue damage. Related to this, although mitochondria are typically thought of in terms of energy production, it has recently become clear that these important organelles also orchestrate the inflammatory response via multiple mechanisms. Dysregulated inflammation is a well-recognised problem in skeletal joint diseases, such as rheumatoid arthritis. Interestingly osteoarthritis (OA), despite traditionally being known as a 'non-inflammatory arthritis', now appears to involve an element of chronic inflammation. OA is considered an umbrella term for a family of diseases stemming from a range of aetiologies (age, obesity etc.), but all with a common presentation. One particular OA sub-set called Post-Traumatic OA (PTOA) results from acute mechanical injury to the joint. Whether the initial mechanical tissue damage, or the subsequent inflammatory response drives disease, is currently unclear. In the former case; mechanobiological properties of cells/tissues in the joint are a crucial consideration. Many such cell-types have been shown to be exquisitely sensitive to their mechanical environment, which can alter their mitochondrial and cellular function. For example, in bone and cartilage cells fluid-flow induced shear stresses can modulate cytoskeletal dynamics and gene expression profiles. More recently, immune cells were shown to be highly sensitive to hydrostatic pressure. In each of these cases mitochondria were central to these responses. In terms of acute inflammation, mitochondria may have a pivotal role in linking joint tissue injury with chronic disease. These processes could involve the immune cells recruited to the joint, native/resident joint cells that have been damaged, or both. Taken together, these observations suggest that mitochondria are likely to play an important role in linking acute joint tissue injury, inflammation, and long-term chronic joint degeneration - and that the process involves mechanobiological factors. In this review, we will explore the links between mechanobiology, mitochondrial function, inflammation/tissue-damage in joint injury and disease. We will also explore some emerging mitochondrial therapeutics and their potential for application in PTOA.

The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells.

Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness-two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity-which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.

Intraarticular injection of liposomal adenosine reduces cartilage damage in established murine and rat models of osteoarthritis.

Osteoarthritis (OA) affects nearly 10% of the population of the United States and other industrialized countries and, at present, short of surgical joint replacement, there is no therapy available that can reverse the progression of the disease. Adenosine, acting at its A2A receptor (A2AR), is a critical autocrine factor for maintenance of cartilage homeostasis and here we report that injection of liposomal suspensions of either adenosine or a selective A2AR agonist, CGS21680, significantly reduced OA cartilage damage in a murine model of obesity-induced OA. The same treatment also improved swelling and preserved cartilage in the affected knees in a rat model of established post-traumatic OA (PTOA). Differential expression analysis of mRNA from chondrocytes harvested from knees of rats with PTOA treated with liposomal A2AR agonist revealed downregulation of genes associated with matrix degradation and upregulation of genes associated with cell proliferation as compared to liposomes alone. Studies in vitro and in affected joints demonstrated that A2AR ligation increased the nuclear P-SMAD2/3/P-SMAD1/5/8 ratio, a change associated with repression of terminal chondrocyte differentiation. These results strongly suggest that targeting the A2AR is an effective approach to treat OA.

Innate Rhythms: Clocks at the Center of Monocyte and Macrophage Function.

The circadian cycle allows organisms to track external time of day and predict/respond to changes in the external environment. In higher order organisms, circadian rhythmicity is a central feature of innate and adaptive immunity. We focus on the role of the molecular clock and circadian rhythmicity specifically in monocytes and macrophages of the innate immune system. These cells display rhythmicity in their internal functions, such as metabolism and inflammatory mediator production as well as their external functions in pathogen sensing, phagocytosis, and migration. These inflammatory mediators are of clinical interest as many are therapeutic targets in inflammatory disease such as cardiovascular disease, diabetes, and rheumatoid arthritis. Moreover, circadian rhythm disruption is closely linked with increased prevalence of these conditions. Therefore, understanding the mechanisms by which circadian disruption affects monocyte/macrophage function will provide insights into novel therapeutic opportunities for these chronic inflammatory diseases.

Immunometabolism around the Clock.

Almost every cell has a molecular clock, which controls gene expression on a 24-h cycle, providing circadian rhythmicity. An example of a circadian behaviour common to most organisms is the feeding/fasting cycle, which shapes whole-body metabolism. However, the exact mechanisms by which the clock controls cellular metabolism have only recently become clear. The molecular clock and related metabolic pathways are also key drivers of immunity. Thus, a natural convergence of circadian biology, metabolism, and immunology has emerged to form a new field that we term 'circadian immunometabolism'. Expanding our understanding of this field will provide insights into chronic conditions such as obesity, cancer, diabetes, cardiovascular disease, and arthritis.

In Vivo Osteocyte Mechanotransduction: Recent Developments and Future Directions.

PURPOSE OF REVIEW: Mechanical loading is an essential stimulus for skeletal tissues. Osteocytes are primarily responsible for sensing mechanical stimuli in bone and for orchestrating subsequent responses. This is critical for maintaining homeostasis, and responding to injury/disease. The osteocyte mechanotransduction pathway, and the downstream effects it mediates, is highly complex. In vivo models have proved invaluable in understanding this process. This review summarizes the commonly used models, as well as more recently developed ones, and describes how they are used to address emerging questions in the field. RECENT FINDINGS: Minimally invasive animal models can be used to determine mechanisms of osteocyte mechanotransduction, at the cell and molecular level, while simultaneously reducing potentially confounding responses such as inflammation/wound-healing. The details of osteocyte mechanotransduction in bone are gradually becoming clearer. In vivo model systems are a key tool in pursing this question. Advances in this field are explored and discussed in this review.

Bone Microdamage in Acute Knee Injury.

PURPOSE OF REVIEW: This review summarizes what is known about how bone tissue responds to microdamage, and how this applies to the subchondral region. This has significant relevance to acute joint injury, and is related to the occurrence of bone marrow lesions (BMLs) which are seen by MRI in 80% of acute knee joint injuries. Here, we review what is known about these phenomena (microcracks and BMLs) in the literature and discuss potential mechanisms by which they may be linked. RECENT FINDINGS: The recent findings in this field have shown that microcracks in bone initiate targeted remodeling via RANKL expression in osteocytes. Other work has shown that subchondral microcracks co-localize with BMLs as viewed by MRI. Finally, BMLs are associated with pain and structural joint degeneration. This paper demonstrates that subchondral microcracks likely occur during acute joint injury, and are closely linked to BML that are seem by clinical MRI and thus are potentially involved in the subsequent joint degeneration that occurs after injury.

Crossed K-Wires Versus Intramedullary Headless Screw Fixation of Unstable Metacarpal Neck Fractures: A Biomechanical Study.

Background: Intramedullary headless screw (IMHS) has shown promise as an alternative to other fixation devices for metacarpal neck fractures. The purpose of this study was to assess the biomechanical performance of IMHS versus the commonly-used crossed K-wire technique. We hypothesized that IMHS fixation provides superior stability to K-wires. Methods: A metacarpal neck fracture model in 23 human cadaveric metacarpals was created. The specimens were divided into two groups based upon fixation method: Group 1, 3 mm intramedullary headless screw; and Group 2, 0.045 inch crossed K-wires. A cantilever bending model was used to assess load-to-failure (LTF), maximum displacement, energy absorption, and stiffness. Results: The mean LTF was 70.6 ± 30.1 N for IMHS and 97.5 ± 34.7 N for crossed K-wires. Mean stiffness was 11.3 ± 3.4 N/mm and 17.7 ± 7.8 N/mm for IMHS and crossed K-wires, respectively. The mean maximum displacement was 20.2 ± 4.6 mm for IMHS and 24.1 ± 3.7 mm for crossed K-wires. Moreover, mean energy absorption was 778.3 ± 528.9 Nmm and 1095.9 ± 454.4 Nmm, respectively, for IMHS and crossed K-wires. Crossed K-wires demonstrated significantly higher stiffness and maximum displacement than IMHS (p < 0.05). Conclusions: IMHS fixation of unstable metacarpal neck fractures offers less stability compared to crossed K-wires when loaded in bending. Clinical Relevance: Crossed K-wires offer superior stability for the treatment of metacarpal neck fractures. These results reveal that IMHS fixation is less favorable biomechanically and should be cautiously selected with regards to fracture stability.

Topical vancomycin and its effect on survival and migration of osteoblasts, fibroblasts, and myoblasts: An in vitro study.

The purpose of this study was to examine the influence of topical vancomycin on cell migration and survival of tissue healing cells. Human osteoblasts, myoblasts and fibroblasts were exposed to vancomycin at concentrations of 1, 3, 6, or 12 mg/cm2 for either a 1-h or 48-h (continuous) duration. Continuous exposure to all vancomycin concentrations significantly reduced cell survival (<22% cells survived) and migration in osteoblasts and myoblasts (P < 0.001). 1-h vancomycin exposure reduced osteoblast and myoblast survival and migration only at 12 mg/cm2 (P < 0.001). Further in vivo studies are warranted to optimize the dosage of intrawound vancomycin.

Bone marrow lesions in osteoarthritis: What lies beneath.

Osteoarthritis (OA) is the most common joint disease in the United States, affecting more than 30 million people, and is characterized by cartilage degeneration in articulating joints. OA can be viewed as a group of overlapping disorders, which result in functional joint failure. However, the precise cellular and molecular events within which lead to these clinically observable changes are neither well understood nor easily measurable. It is now clear that multiple factors, in multiple joint tissues, contribute to degeneration. Changes in subchondral bone are recognized as a hallmark of OA, but are normally associated with late-stage disease when degeneration is well established. However, early changes such as Bone Marrow Lesions (BMLs) in OA are a relatively recent discovery. BMLs are patterns from magnetic resonance images (MRI) that have been linked with pain and cartilage degeneration. Their potential utility in predicting progression, or as a target for therapy, is not yet fully understood. Here, we will review the current state-of-the-art in this field under three broad headings: (i) BMLs in symptomatic OA: malalignment, joint pain, and disease progression; (ii) biological considerations for bone-cartilage crosstalk in joint disease; and (iii) mechanical factors that may underlie BMLs and drive their communication with other joint tissues. Thus, this review will provide insights on this topic from a clinical, biological, and mechanical perspective. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1818-1825, 2018.

The Effect of Growth Hormone on Chondral Defect Repair.

Objective Focal chondral defects alter joint mechanics and cause pain and debilitation. Microfracture is a surgical technique used to treat such defects. This technique involves penetration of subchondral bone to release progenitor cells and growth factors from the marrow to promote cartilage regeneration. Often this results in fibrocartilage formation rather than structured hyaline cartilage. Some reports have suggested use of growth hormone (GH) with microfracture to augment cartilage regeneration. Our objective was to test whether intra-articular (IA) GH in conjunction with microfracture, improves cartilage repair in a rabbit chondral defect model. We hypothesized that GH would exhibit a dose-dependent improvement in regeneration. Design Sixteen New Zealand white rabbits received bilateral femoral chondral defects and standardized microfracture repair. One group of animals ( n = 8) received low-dose GH by IA injection in the left knee, and the other group ( n = 8) received high-dose GH in the same manner. All animals received IA injection of saline in the contralateral knee as control. Serum assays, macroscopic grading, and histological analyses were used to assess any improvements in cartilage repair. Results Peripheral serum GH was not elevated postoperatively ( P = 0.21). There was no improvement in macroscopic grading scores among either of the GH dosages ( P = 0.83). Scoring of safranin-O-stained sections showed no improvement in cartilage regeneration and some evidence of increased bone formation in the GH-treated knees. Conclusions Treatment with either low- or high-dose IA GH does not appear to enhance short-term repair in a rabbit chondral defect model.

A Biomechanical Study of Two Distinct Methods of Anterior Cruciate Ligament Rupture, and a Novel Surgical Reconstruction Technique, in a Small Animal Model of Posttraumatic Osteoarthritis.

Small animal models are critical for studies of sports-related knee injury and disease such as posttraumatic osteoarthritis (PTOA) following anterior cruciate ligament (ACL) rupture. In such models, ACL damage can be achieved by surgical transection or, using a more recent innovation, by noninvasive biomechanical means. Whether these approaches differentially alter normal mechanics is unknown. Furthermore, while surgical reconstruction of ruptured ACL can greatly improve joint stability, its effect on PTOA development is also unclear. Our primary purpose was to characterize rodent knee joint mechanics in two models of ACL rupture using a novel quantitative laxity mechanical test. Our secondary aim was to characterize a new reconstruction technique using autograft tail tendon, and to assess its effect on joint mechanics. Our hypothesis was that surgical ACL transection would have a greater effect on joint mechanics. A total of 24 rat knee specimens underwent surgical or biomechanical ACL rupture and were stabilized using a new reconstruction technique using autograft tail tendon. Joint mechanics were assessed three times; preinjury, postinjury, and again after reconstruction, using quantitative joint laxity testing. Primary test readouts were maximum anteroposterior (AP) laxity, loading curve slope, and energy absorption. Student's t-tests were performed to identify intragroup differences. All surgical transections were completed successfully; maximum load in the biomechanical model was 67 ± 7.7 N, with a coefficient of variation of 11.43%. Surgical transection caused increased AP laxity, while biomechanical injury nonsignificantly increased this parameter. In both cases, these changes recovered to baseline by reconstruction. Loading curve slope was reduced in both models and was also returned to baseline by repair. Energy absorption followed the same pattern except it remained significantly different from baseline postreconstruction in the surgical group. This study supports our hypothesis knee joint mechanics is differentially affected by injury mechanism in a small animal model. We also report a novel reconstruction technique in this model, using autograft tail tendon.

Progranulin derivative Atsttrin protects against early osteoarthritis in mouse and rat models.

BACKGROUND: Atsttrin, an engineered protein composed of three tumor necrosis factor receptor (TNFR)-binding fragments of progranulin (PGRN), shows therapeutic effect in multiple murine models of inflammatory arthritis . Additionally, intra-articular delivery of PGRN protects against osteoarthritis (OA) progression. The purpose of this study is to determine whether Atsttrin also has therapeutic effects in OA and the molecular mechanisms involved. METHODS: Surgically induced and noninvasive rupture OA models were established in mouse and rat, respectively. Cartilage degradation and OA were evaluated using Safranin O staining, immunohistochemistry, and ELISA. Additionally, expressions of pain-related markers, degenerative factors, and anabolic and catabolic markers known to be involved in OA were analyzed. Furthermore, the anabolic and anti-catabolic effects and underlying mechanisms of Atsttrin were determined using in-vitro assays with primary chondrocytes. RESULTS: Herein, we found Atsttrin effectively prevented the accelerated OA phenotype associated with PGRN deficiency. Additionally, Atsttrin exhibited a preventative effect in OA by protecting articular cartilage and reducing OA-associated pain in both nonsurgically induced rat and surgically induced murine OA models. Mechanistic studies revealed that Atsttrin stimulated TNFR2-Akt-Erk1/2-dependent chondrocyte anabolism, while inhibiting TNFα/TNFR1-mediated inflammatory catabolism. CONCLUSIONS: These findings not only provide new insights into the role of PGRN and its derived engineered protein Atsttrin in cartilage homeostasis as well as OA in vivo, but may also lead to new therapeutic alternatives for OA as well as other relative degenerative joint diseases.

Endogenous adenosine maintains cartilage homeostasis and exogenous adenosine inhibits osteoarthritis progression.

Osteoarthritis (OA) is characterized by cartilage destruction and chondrocytes have a central role in this process. With age and inflammation chondrocytes have reduced capacity to synthesize and maintain ATP, a molecule important for cartilage homeostasis. Here we show that concentrations of ATP and adenosine, its metabolite, fall after treatment of mouse chondrocytes and rat tibia explants with IL-1ß, an inflammatory mediator thought to participate in OA pathogenesis. Mice lacking A2A adenosine receptor (A2AR) or ecto-5'nucleotidase (an enzyme that converts extracellular AMP to adenosine) develop spontaneous OA and chondrocytes lacking A2AR develop an 'OA phenotype' with increased expression of Mmp13 and Col10a1. Adenosine replacement by intra-articular injection of liposomal suspensions containing adenosine prevents development of OA in rats. These results support the hypothesis that maintaining extracellular adenosine levels is an important homeostatic mechanism, loss of which contributes to the development of OA; targeting adenosine A2A receptors might treat or prevent OA.

Automated Bone Segmentation and Surface Evaluation of a Small Animal Model of Post-Traumatic Osteoarthritis.

MicroCT imaging allows for noninvasive microstructural evaluation of mineralized bone tissue, and is essential in studies of small animal models of bone and joint diseases. Automatic segmentation and evaluation of articular surfaces is challenging. Here, we present a novel method to create knee joint surface models, for the evaluation of PTOA-related joint changes in the rat using an atlas-based diffeomorphic registration to automatically isolate bone from surrounding tissues. As validation, two independent raters manually segment datasets and the resulting segmentations were compared to our novel automatic segmentation process. Data were evaluated using label map volumes, overlap metrics, Euclidean distance mapping, and a time trial. Intraclass correlation coefficients were calculated to compare methods, and were greater than 0.90. Total overlap, union overlap, and mean overlap were calculated to compare the automatic and manual methods and ranged from 0.85 to 0.99. A Euclidean distance comparison was also performed and showed no measurable difference between manual and automatic segmentations. Furthermore, our new method was 18 times faster than manual segmentation. Overall, this study describes a reliable, accurate, and automatic segmentation method for mineralized knee structures from microCT images, and will allow for efficient assessment of bony changes in small animal models of PTOA.

Microdamage induced by in vivo Reference Point Indentation in mice is repaired by osteocyte-apoptosis mediated remodeling.

Reference Point Indentation (RPI) is a technology that is designed to measure mechanical properties that relate to bone toughness, or its ability to resist crack growth, in vivo. Independent of the mechanical parameters generated by RPI, its ability to initiate and propagate microcracks in bone is itself an interesting issue. Microcracks have a crucial biological relevance in bone, are central to its ability to maintain homeostasis. In healthy tissues, a process of targeted remodeling routinely repairs microcracks in a process mediated by osteocyte apoptosis. However, in diseases such as osteoporosis this process becomes deficient and microcracks can accumulate. Small animal models such are crucial for the study of such diseases, but it is technically challenging to create microcracks in these animals without causing outright failure. Therefore we sought to use RPI as a focal microdamage placement tool, to introduce microcracks to mouse long bones and investigate whether the same pathway mediates their repair as that described in other microdamage systems. We first used SEM to confirm that microdamage is formed RPI in mouse bone. Then, since RPI is carried out transdermally, we sought to confirm that no periosteal response occurred at the indented region. We then used a pan-caspase inhibitor (QVD) to determine whether osteocyte apoptosis plays the same pivotal role in microdamage repair in this model, as has been demonstrated in others. In conclusion, we validated that the microdamage-apoptosis-remodeling pathway is maintained with this method of microdamage induction in mice. We show that RPI can be used as a reliable and reproducible microdamage placement tool in living mouse long bones without inducing a periosteal response. We also used a caspase inhibitor, to block osteocyte apoptosis and thus abrogate the remodeling response to microdamage. This demonstrates that the well described microdamage repair system, involving targeted remodeling mediated by osteocyte apoptosis, is conserved in this novel mouse model using an in vivo RPI loading system.