Local delivery of FTY720 induces neutrophil activation through chemokine signaling in an oronasal fistula model.

PURPOSE: Cleft palate repair surgeries lack a regenerative reconstructive option and, in many cases, develop complications including oronasal fistula (ONF). Our group has developed a novel murine phenocopy of ONF to study the oral cavity wound healing program. Using this model, our team previously identified that delivery of FTY720 on a nanofiber scaffold had a unique immunomodulatory effect directing macrophages and monocytes into a pro-regenerative state during ONF healing. Here, the objective of this study was to determine the effects of local biomaterial-based FTY720 delivery in the ONF model on the early bulk gene expression and neutrophil phenotypic response within the regenerating tissue. METHODS: Using a mouse model of ONF formation, a palate defect was created and was treated with FTY720 nanofiber scaffolds or (blank) vehicle control nanofibers. At 1 and 3 days post-implantation, ONF oral mucosal tissue from the defect region was collected for RNA sequencing analysis or flow cytometry. For the RNA-seq expression profiling, intracellular pathways were assessed using the KEGG Pathway database and Gene Ontology (GO) Terms enrichment interactive graph. To assess the effects of FTY720 on different neutrophil subpopulations, flow cytometry data was analyzed using pseudotime analysis based on Spanning-tree Progression Analysis of Density-normalized Events (SPADE). RESULTS: RNA sequencing analysis of palate mucosa injured tissue identified 669 genes that were differentially expressed (DE) during the first 3 days of ONF wound healing after local delivery of FTY720, including multiple genes in the sphingolipid signaling pathway. Evaluation of the DE genes at the KEGG Pathway database also identified the inflammatory immune response pathways (chemokine signaling, cytokine-cytokine receptor interaction, and leukocyte transendothelial migration), and the Gene Ontology enrichment analysis identified neutrophil chemotaxis and migration terms. SPADE dendrograms of CD11b+Ly6G+ neutrophils at both day 1 and day 3 post-injury showed significantly distinct subpopulations of neutrophils in oral mucosal defect tissue from the FTY720 scaffold treatment group compared to the vehicle control group (blank). Increased expression of CD88 and Vav1, among other genes, were found and staining of the ONF area demonstrated increased VAV1 staining in FTY720-treated healing oral mucosa. CONCLUSION: Treatment of oral mucosal defects using FTY720 scaffolds is a promising new immunotherapy to improve healing outcomes and reducing ONF formation during cleft palate surgical repair. Local delivery of FTY720 nanofiber scaffolds during ONF healing significantly shifted early gene transcription associated with immune cell recruitment and modulation of the immune microenvironment results in distinct neutrophil subpopulations in the oral mucosal defect tissue that provides a critical shift toward pro-regenerative immune signaling. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40883-021-00208-z.

Extracellular matrix compression temporally regulates microvascular angiogenesis.

Mechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Previously, we found that dynamic matrix compression in vivo potently regulated revascularization during bone tissue regeneration; however, whether matrix deformations directly regulate angiogenesis remained unknown. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Immediate load initiation inhibited angiogenesis and expression of early sprout tip cell selection genes, while delayed loading enhanced microvascular network formation and upstream signaling pathways. This research provides foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

Polyvinylpyrolidone-functionalized silver nanoparticles do not affect aerobic performance or fractional rates of protein synthesis in rainbow trout (Oncorhynchus mykiss).

Aerobic performance in fish is linked to individual and population fitness and can be impacted by anthropogenic contaminants. Exposure to some engineered nanomaterials, including silver nanoparticles (nAg), reduces rates of oxygen consumption in some fish species, but the underlying mechanisms remain unclear. In addition, their effects on swim performance have not been studied. Our aim was to quantify the impact of exposure to functionalized nAg on aerobic scope and swim performance in rainbow trout (Oncorhychus mykiss) and to characterize the contribution of changing rates of protein synthesis to these physiological endpoints. Fish were exposed for 48 h to 5 nm polyvinylpyrolidone-functionalized nAg (nAgPVP; 100 µg L-1) or 0.22 µg L-1 Ag+ (as AgNO3), which was the measured quantity of Ag released from the nAgPVP over that time period. Aerobic scope, critical swimming speed (Ucrit), and fractional rates of protein synthesis (Ks), were then assessed, along with indicators of osmoregulation and cardiotoxicity. Neither nAgPVP, nor Ag+ exposure significantly altered aerobic scope, its component parts, or swim performance. Ks was similarly unaffected in 8 tissue types, though it tended to be lower in liver of nAgPVP treated fish. The treatments tended to decrease gill Na+/K+-ATPase activity, but effects were not significant. The latter results suggest that a longer or more concentrated nAgPVP exposure may induce significant effects. Although this same formulation of nAgPVP is bioactive in other fish, it had no effects on rainbow trout under the conditions tested. Such findings on common model animals like trout may thus misrepresent the safety of nAg to more sensitive species.

Effects of treadmill running and limb immobilization on knee cartilage degeneration and locomotor joint kinematics in rats following knee meniscal transection.

OBJECTIVE: This study examined the effects of reduced and elevated weight bearing on post-traumatic osteoarthritis (PTOA) development, locomotor joint kinematics, and degree of voluntary activity in rats following medial meniscal transection (MMT). DESIGN: Twenty-one adult rats were subjected to MMT surgery of the left hindlimb and then assigned to one of three groups: (1) regular (i.e., no intervention), (2) hindlimb immobilization, or (3) treadmill running. Sham surgery was performed in four additional rats. Voluntary wheel run time/distance was measured, and 3D hindlimb kinematics were quantified during treadmill locomotion using biplanar radiography. Rats were euthanized 8 weeks after MMT or sham surgery, and the microstructure of the tibial cartilage and subchondral bone was quantified using contrast enhanced micro-CT. RESULTS: All three MMT groups showed signs of PTOA (full-thickness lesions and/or increased cartilage volume) compared to the sham group, however the regular and treadmill-running groups had greater osteophyte formation than the immobilization group. For the immobilization group, increased volume was only observed in the anterior region of the cartilage. The treadmill-running group demonstrated a greater knee varus angle at mid-stance than the sham group, while the immobilization group demonstrated greater reduction in voluntary running than all the other groups at 2 weeks post-surgery. CONCLUSIONS: Elevated weight-bearing via treadmill running at a slow/moderate speed did not accelerate PTOA in MMT rats when compared to regular weight-bearing. Reduced weight-bearing via immobilization may attenuate overall PTOA but still resulted in regional cartilage degeneration. Overall, there were minimal differences in hindlimb kinematics and voluntary running between MMT and sham rats.

Therapeutic efficacy of intra-articular delivery of encapsulated human mesenchymal stem cells on early stage osteoarthritis.

Mesenchymal stem cells (MSCs) represent a great therapeutic promise in pre-clinical models of osteoarthritis (OA), but many questions remain as to their therapeutic mechanism of action: engraftment versus paracrine action. Encapsulation of human MSCs (hMSCs) in sodium alginate microspheres allowed for the paracrine signaling properties of these cells to be isolated and studied independently of direct cellular engraftment. The objective of the present study was to quantitatively assess the efficacy of encapsulated hMSCs as a disease-modifying therapeutic for OA, using a medial meniscal tear (MMT) rat model. It was hypothesized that encapsulated hMSCs would have a therapeutic effect, through paracrine-mediated action, on early OA development. Lewis rats underwent MMT surgery to induce OA. 1 d post-surgery, rats received intra-articular injections of encapsulated hMSCs or controls (i.e., saline, empty capsules, non-encapsulated hMSCs). Microstructural changes in the knee joint were quantified using equilibrium partitioning of a ionic contrast agent based micro-computed tomography (EPIC-µCT) at 3 weeks post-surgery, an established time point for early OA. Encapsulated hMSCs significantly attenuated MMT-induced increases in articular cartilage swelling and surface roughness and augmented cartilaginous and mineralized osteophyte volumes. Cellular encapsulation allowed to isolate the hMSC paracrine signaling effects and demonstrated that hMSCs could exert a chondroprotective therapeutic role on early stage OA through paracrine signaling alone. In addition to this chondroprotective role, encapsulated hMSCs augmented the compensatory increases in osteophyte formation. The latter should be taken into strong consideration as many clinical trials using MSCs for OA are currently ongoing.

Localized SDF-1α Delivery Increases Pro-Healing Bone Marrow-Derived Cells in the Supraspinatus Muscle Following Severe Rotator Cuff Injury.

To examine how the chemotactic agent stromal cell-derived factor-1alpha (SDF-1α) modulates the unique cellular milieu within rotator cuff muscle following tendon injury, we developed an injectable, heparin-based microparticle platform to locally present SDF-1α within the supraspinatus muscle following severe rotator cuff injury. SDF-1α loaded, degradable, N-desulfated heparin-based microparticles were fabricated, injected into a rat model of severe rotator cuff injury, and were retained for up to 7 days at the site. The resultant inflammatory cell and mesenchymal stem cell populations were analyzed compared to uninjured contralateral controls and, after 7 days, the fold-change in anti-inflammatory, M2-like macrophages (CD11b+CD68+CD163+, 4.3X fold-change) and mesenchymal stem cells (CD29+CD44+CD90+, 3.0X, respectively) was significantly greater in muscles treated with SDF-1α loaded microparticles than unloaded microparticles or injury alone. Our results indicate that SDF-1α loaded microparticles may be a novel approach to shift the cellular composition within the supraspinatus muscle and create a more pro-regenerative milieu, which may provide a platform to improve muscle repair following rotator cuff injury in the future.

Contrast enhanced µCT imaging of early articular changes in a pre-clinical model of osteoarthritis.

OBJECTIVE: The objective of this study was to characterize early osteoarthritis (OA) development in cartilage and bone tissues in the rat medial meniscus transection (MMT) model using non-destructive equilibrium partitioning of an ionic contrast agent micro-computed tomography (EPIC-µCT) imaging. Cartilage fibrillation, one of the first physiological developments in OA, was quantified in the rat tibial plateau as three-dimensional (3D) cartilage surface roughness using a custom surface-rendering algorithm. METHODS: Male Lewis rats underwent MMT or sham-operation in the left leg. At 1- and 3-weeks post-surgery, the animals (n = 7-8 per group) were euthanized and the left legs were scanned using EPIC-µCT imaging to quantify cartilage and bone parameters. In addition, a custom algorithm was developed to measure the roughness of 3D surfaces. This algorithm was validated and used to quantify cartilage surface roughness changes as a function of time post-surgery. RESULTS: MMT surgery resulted in significantly greater cartilage damage and subchondral bone sclerosis with the damage increasing in both severity and area from 1- to 3-weeks post-surgery. Analysis of rendered 3D surfaces could accurately distinguish early changes in joints developing OA, detecting significant increases of 45% and 124% in surface roughness at 1- and 3-weeks post-surgery respectively. CONCLUSION: Disease progression in the MMT model progresses sequentially through changes in the cartilage articular surface, extracellular matrix composition, and then osteophyte mineralization and subchondral bone sclerosis. Cartilage surface roughness is a quantitative, early indicator of degenerative joint disease in small animal OA models and can potentially be used to evaluate therapeutic strategies.

Quantitative pre-clinical screening of therapeutics for joint diseases using contrast enhanced micro-computed tomography.

OBJECTIVE: The development of effective therapies for cartilage protection has been limited by a lack of efficient quantitative cartilage imaging modalities in pre-clinical in vivo models. Our objectives were two-fold: first, to validate a new contrast-enhanced 3D imaging analysis technique, equilibrium partitioning of an ionic contrast agent-micro computed tomography (EPIC-µCT), in a rat medial meniscal transection (MMT) osteoarthritis (OA) model; and second, to quantitatively assess the sensitivity of EPIC-µCT to detect the effects of matrix metalloproteinase inhibitor (MMPi) therapy on cartilage degeneration. METHODS: Rats underwent MMT surgery and tissues were harvested at 1, 2, and 3 weeks post-surgery or rats received an MMPi or vehicle treatment and tissues harvested 3 weeks post-surgery. Parameters of disease progression were evaluated using histopathology and EPIC-µCT. Correlations and power analyses were performed to compare the techniques. RESULTS: EPIC-µCT was shown to provide simultaneous 3D quantification of multiple parameters, including cartilage degeneration and osteophyte formation. In MMT animals treated with MMPi, OA progression was attenuated, as measured by 3D parameters such as lesion volume and osteophyte size. A post-hoc power analysis showed that 3D parameters for EPIC-µCT were more sensitive than 2D parameters requiring fewer animals to detect a therapeutic effect of MMPi. 2D parameters were comparable between EPIC-µCT and histopathology. CONCLUSION: This study demonstrated that EPIC-µCT has high sensitivity to provide 3D structural and compositional measurements of cartilage and bone in the joint. EPIC-µCT can be used in combination with histology to provide a comprehensive analysis to screen new potential therapies.

Localized 3D analysis of cartilage composition and morphology in small animal models of joint degeneration.

OBJECTIVE: Current histological scoring methods to evaluate efficacy of potential therapeutics for slowing or preventing joint degeneration are time-consuming and semi-quantitative in nature. Hence, there is a need to develop and standardize quantitative outcome measures to define sensitive metrics for studying potential therapeutics. The objectives of this study were to use equilibrium partitioning of an ionic contrast agent via Equilibrium Partitioning of an Ionic Contrast-Microcomputed tomography (EPIC-µCT) to quantitatively characterize morphological and compositional changes in the tibial articular cartilage in two distinct models of joint degeneration and define localized regions of interest to detect degenerative cartilage changes. MATERIALS AND METHODS: The monosodium iodoacetate (MIA) and medial meniscal transection (MMT) rat models were used in this study. Three weeks post-surgery, tibiae were analyzed using EPIC-µCT and histology. EPIC-µCT allowed measurement of 3D morphological changes in cartilage thickness, volume and composition. RESULTS: Extensive cartilage degeneration was observed throughout the joint in the MIA model after 3 weeks. In contrast, the MMT model showed more localized degeneration with regional thickening of the medial tibial plateau and a decrease in attenuation consistent with proteoglycan (PG) depletion. Focal lesions were also observed and 3D volume calculated as an additional outcome metric. CONCLUSIONS: EPIC-µCT was used to quantitatively assess joint degeneration in two distinct preclinical models. The MMT model showed similar features to human Osteoarthritis (OA), including localized lesion formation and PG loss, while the MIA model displayed extensive cartilage degeneration throughout the joint. EPIC-µCT imaging provides a rapid and quantitative screening tool for preclinical evaluation of OA therapeutics.