Thermoresponsive Elastin-b-Collagen-Like Peptide Bioconjugate Nanovesicles for Targeted Drug Delivery to Collagen-Containing Matrices.

Over the past few decades, (poly)peptide block copolymers have been widely employed in generating well-defined nanostructures as vehicles for targeted drug delivery applications. We previously reported the assembly of thermoresponsive nanoscale vesicles from an elastin-b-collagen-like peptide (ELP-CLP). The vesicles were observed to dissociate at elevated temperatures, despite the LCST-like behavior of the tethered ELP domain, which is suggested to be triggered by the unfolding of the CLP domain. Here, the potential of using the vesicles as drug delivery vehicles for targeting collagen-containing matrices is evaluated. The sustained release of an encapsulated model drug was achieved over a period of 3 weeks, following which complete release could be triggered via heating. The ELP-CLP vesicles show strong retention on a collagen substrate, presumably through collagen triple helix interactions. Cell viability and proliferation studies using fibroblasts and chondrocytes suggest that the vesicles are highly cytocompatible. Additionally, essentially no activation of a macrophage-like cell line is observed, suggesting that the vesicles do not initiate an inflammatory response. Endowed with thermally controlled delivery, the ability to bind collagen, and excellent cytocompatibility, these ELP-CLP nanovesicles are suggested to have significant potential in the controlled delivery of drugs to collagen-containing matrices and tissues.

Family Matters: Examining Child Abuse and Neglect as Family Dysfunction for Minority Youth Living in Extreme Poverty.

Two competing models of child abuse and neglect (scapegoat vs. family dysfunction) are used to illustrate how the specification of victims ("index" victim vs. all children in household) from incidents of child abuse and neglect can be used to improve estimates of maltreatment for at-risk minority youth. Child Protection Services records were searched in 2005 for 366 "index" victims who were surveyed for 5 consecutive years (from 1998 to 2002) for the Mobile Youth Survey as well as other siblings in the household. The findings indicate that the baseline estimate of any maltreatment, sexual abuse, physical abuse, and neglect increased by 68%, 26%, 33%, and 74%, respectively, after adjusting for incidents that involved multiple victims (i.e., maltreatment as family dysfunction). In addition, the baseline estimate of more severe (indicated) incidents of physical abuse and neglect increased by 67% and 64%, respectively, after accounting for incidents that involved multiple victims, but there were no incidents of more severe (indicated) sexual abuse that involved multiple victims. Similarly, baseline estimates of age of onset (or chronicity) of maltreatment during childhood and adolescence increased by 62% and 26%, respectively. Baseline estimates for youth with 3 or more years of maltreatment and youth with 3 or more incidents of maltreatment both increased by about 71%. The implications of these findings for policy and practice as well as areas for future research are also discussed.

Pleiotropic Effects of Simvastatin and Losartan in Preclinical Models of Post-Traumatic Elbow Contracture.

Elbow trauma can lead to post-traumatic joint contracture (PTJC), which is characterized by loss of motion associated with capsule/ligament fibrosis and cartilage damage. Unfortunately, current therapies are often unsuccessful or cause complications. This study aimed to determine the effects of prophylactically administered simvastatin (SV) and losartan (LS) in two preclinical models of elbow PTJC: an in vivo elbow-specific rat injury model and an in vitro collagen gel contraction assay. The in vivo elbow rat (n = 3-10/group) injury model evaluated the effects of orally administered SV and LS at two dosing strategies [i.e., low dose/high frequency/short duration (D1) vs. high dose/low frequency/long duration (D2)] on post-mortem elbow range of motion (via biomechanical testing) as well as capsule fibrosis and cartilage damage (via histopathology). The in vitro gel contraction assay coupled with live/dead staining (n = 3-19/group) evaluated the effects of SV and LS at various concentrations (i.e., 1, 10, 100 µM) and durations (i.e., continuous, short, or delayed) on the contractibility and viability of fibroblasts/myofibroblasts [i.e., NIH3T3 fibroblasts with endogenous transforming growth factor-beta 1 (TGFß1)]. In vivo, no drug strategy prevented elbow contracture biomechanically. Histologically, only SV-D2 modestly reduced capsule fibrosis but maintained elevated cellularity and tissue hypertrophy, and both SV strategies lessened cartilage damage. SV modest benefits were localized to the anterior region, not the posterior, of the joint. Neither LS strategy had meaningful benefits in capsule nor cartilage. In vitro, irrespective of the presence of TGFß1, SV (≥10 µM) prevented gel contraction partly by decreasing cell viability (100 µM). In contrast, LS did not prevent gel contraction or affect cell viability. This study demonstrates that SV, but not LS, might be suitable prophylactic drug therapy in two preclinical models of elbow PTJC. Results provide initial insight to guide future preclinical studies aimed at preventing or mitigating elbow PTJC.

Preclinical Models of Elbow Injury and Pathology.

The human elbow is a complex joint that is essential for activities of daily living requiring the upper extremities; however, this complexity generates significant challenges when considering its response to injury and management of treatment. The current understanding of elbow injury and pathologies lags behind that of other joints and musculoskeletal tissues. Most research on the elbow joint is mainly focused on the late-stage disease states when irreversible damage has occurred. Consequentially, the specific contribution and relative time course of different elbow tissues in disease progression, as well as optimized approaches for treating such conditions, remains largely unknown. Given the challenge of studying elbow pathologies in humans, preclinical models can serve as ideal alternatives. However, a limited number of preclinical models exist to investigate elbow injury and pathology. This review highlights significant clinical elbow diseases and the preclinical models currently available to recapitulate these diseases, while also providing recommendations for the development of future preclinical models. Overall, this review will serve as a guide for preclinical models studying injuries and pathologies of the elbow, with the long-term goal of developing novel intervention strategies to improve the treatment of elbow diseases in human patients.

Early, focal changes in cartilage cellularity and structure following surgically induced meniscal destabilization in the mouse.

Post-traumatic osteoarthritis (PTOA) is an accelerated form of osteoarthritic cartilage degeneration affecting approximately 20-50% of patients experiencing joint injury. Currently PTOA is incurable; to better understand the etiology of PTOA and to develop rational anti-osteoarthritic therapies, it is critical to understand the spatiotemporal initiation and the progression of PTOA. In this study, we employed semi-quantitative histological scoring and quantitative damage analysis to examine disease progression in the murine destabilization of the medial meniscus (DMM) model of PTOA from early (3 days) through late- (112 days) disease timepoints. We observed significant, progressive articular cartilage (AC) cellular, and structural changes in the medial compartments of injured joints as early as 3 days. Spatially within the joint, cartilage damage (erosions) were observed anteriorly at 84 days. Furthermore, a drastic loss in chondrocyte number (by 3 days), surface damage (at 7 days), and cartilage erosion (at 84 days) was found to co-localize to the specific region of the medial tibial plateau AC that experienced a change in meniscal coverage due to meniscal extrusion following DMM. Taken together, these results suggest that DMM-mediated extrusion of the medial meniscus leads to rapid, spatially dependent changes in AC cellularity and structure, and precipitates the focal degeneration of cartilage associated with PTOA. Importantly, this study suggests that joint instability injuries may trigger immediate (<3 days) processes within a small population of chondrocytes that directs the initiation and progression of PTOA, and that development of chondroprotective strategies for preventing and/or delaying PTOA-related cartilage degeneration are best targeted toward these immediately early processes following joint injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:537-547, 2017.

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing.

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.

Effects of Osmolarity on the Spontaneous Calcium Signaling of In Situ Juvenile and Adult Articular Chondrocytes.

Calcium is a universal second messenger that mediates the metabolic activity of chondrocytes in articular cartilage. Spontaneous intracellular calcium ([Ca(2+)]i) oscillations, similar to those in neurons and myocytes, have recently been observed in chondrocytes. This study analyzed and compared the effects of different osmotic environments (hypertonic, hypotonic, and isotonic) on the spontaneous [Ca(2+)]i signaling of in situ chondrocytes residing in juvenile and adult cartilage explants. In spite of a lower cell density, a significantly higher percentage of chondrocytes in adult cartilage under all osmotic environments demonstrated spontaneous [Ca(2+)]i oscillations than chondrocytes in juvenile cartilage. For both juvenile and adult chondrocytes, hypotonic stress increased while hypertonic stress decreased the response rates. Furthermore, the spatiotemporal characteristics of the [Ca(2+)]i peaks vary in an age-dependent manner. In the hypotonic environment, the [Ca(2+)]i oscillation frequency of responsive adult cells is almost tripled whereas the juvenile cells respond with an increased duration and magnitude of each [Ca(2+)]i peak. Both juvenile and adult chondrocytes demonstrated significantly slower [Ca(2+)]i oscillations with longer rising and recovery time under the hypertonic condition. Taken together, these results shed new insights into the interplay between age and osmotic environment that may regulate the fundamental metabolism of chondrocytes.

Tendon repair is compromised in a high fat diet-induced mouse model of obesity and type 2 diabetes.

INTRODUCTION: The obesity epidemic has resulted in a large increase in type 2 diabetes (T2D). While some secondary complications of T2D are well recognized and their cellular and molecular mechanisms are defined, the impact of T2D on the musculoskeletal system is less understood. Clinical evidence suggests that tendon strength and repair are compromised. Here, a mouse model of obesity and T2D recapitulates the deleterious effects of this condition on tendon repair. METHODS: Male C57BL/6J mice at 5 weeks of age were placed on a high fat (HF)(60% kcal) or low fat (10% kcal) diet for 12 weeks. The flexor digitorum longus (FDL) tendon was then injured by puncturing it with a beveled needle. Progression of FDL tendon healing was assessed through biomechanical and histological analysis at 0, 7, 14 and 28 days post-injury. RESULTS: HF-fed mice displayed increased body weight and elevated fasting glucose levels, both consistent with T2D. No differences in biomechanical properties of the uninjured FDL tendon were observed after 12 weeks on HF versus lean diets, but decreased maximum force in uninjured tendons from HF-fed mice was observed at 24 weeks. Following puncture injury, tendons from HF-fed mice displayed impaired biomechanical properties at day 28 post injury. In support of defective repair in the HF-fed mice, histological examination of the injury site showed a smaller area of repair and lower cell content in the repair area of HF-fed mice. Insulin receptors were expressed in most cells at the injury site regardless of diet. DISCUSSION: The HF-diet mouse model of obesity and T2D reproduces the impaired tendon healing that is observed in this patient population. The exact mechanism is unknown, but we hypothesize that a cellular defect, perhaps involving insulin resistance, leads to decreased proliferation or recruitment to the injury site, and ultimately contributes to defective tendon healing.