Repair Phase Research Articles

The Spectrum of CT Findings of COVID-19 Pneumonia: Acute Alveolar Insult and Organizing Pneumonia as Different Phases of Lung Injury and Repair.

PurposeTo analyze the findings and serial changes in chest CT lesions in 123 symptomatic patients with coronavirus disease 2019 (COVID-19).Materials and MethodsFrom February 19 to April 7, 2020, a total of 123 confirmed COVID-19 patients (male, 44; female, 79; mean age, 59.2 ± 18.6) were enrolled in this retrospective study. A total of 234 CT scans were reviewed for the following patterns: acute alveolar insult (AAI) patterns: ground-glass opacity (GGO), crazy-paving appearance, mixed pattern, and consolidation; organizing pneumonia (OP) patterns: perilobular patterns, band opacity, curvilinear opacity, reversed halo opacity, and small nodular consolidation; resolving patterns: pure GGO, remnant curvilinear, small nodular consolidation, and serial changes of lung abnormalities. We compared the proportions of AAI pattern, OP pattern, or resolving pattern with time progression and analyzed the association between the patterns and disease severity using Pearson chi-square and Fisher's exact test.ResultsPredominant CT patterns were AAI pattern (87%) in the early hospital period group (0–10 days, after the onset of symptoms), OP pattern (45.7%) in the later hospital period group (after 10 days), and resolving pattern in discharge and follow-up group (47.2% and 84.8%, respectively). The difference in the proportions of predominant CT patterns with time progression was statistically significant (p < 0.001, Pearson's chi-square test). No statistically significant association was observed between the patterns and disease severity (p = 0.055, Fisher's exact test). No fibrous changes in the lesions were observed on follow-up CT scans.ConclusionThe serial CT scans of COVID-19 patients showed the spectrum of COVID pneumonia CT manifestations as different phases of lung injury and repair.

Potential role of myeloid-derived suppressor cells in transition from reaction to repair phase of bone healing process.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with immunosuppressive functions; these cells play a key role in infection, immunization, chronic inflammation, and cancer. Recent studies have reported that immunosuppression plays an important role in the healing process of tissues and that Treg play an important role in fracture healing. MDSCs suppress active T cell proliferation and reduce the severity of arthritis in mice and humans. Together, these findings suggest that MDSCs play a role in bone biotransformation. In the present study, we examined the role of MDSCs in the bone healing process by creating a bone injury at the tibial epiphysis in mice. MDSCs were identified by CD11b and GR1 immunohistochemistry and their role in new bone formation was observed by detection of Runx2 and osteocalcin expression. Significant numbers of MDSCs were observed in transitional areas from the reactionary to repair stages. Interestingly, MDSCs exhibited Runx2 and osteocalcin expression in the transitional area but not in the reactionary area. And at the same area, cllagene-1 and ALP expression level increased in osteoblast progenitor cells. These data is suggesting that MDSCs emerge to suppress inflammation and support new bone formation. Here, we report, for the first time (to our knowledge), the role of MDSCs in the initiation of bone formation. MDSC appeared at the transition from inflammation to bone making and regulates bone healing by suppressing inflammation.

Recovery of Liver Sinusoidal Endothelial Cells Following Monocrotaline-induced Liver Injury.

Although the pathology of sinusoidal obstruction syndrome (SOS) is characterized by damage to liver sinusoidal endothelial cells (LSECs), the processes underlying LSEC repair are incompletely understood. The angiopoietin (Ang)/Tie system contributes to angiogenesis. The present study aimed to examine the processes of LSEC repair and the involvement of the Ang/Tie pathway in LSEC recovery. Experimentally, SOS was induced by intraperitoneal injection of monocrotaline (MCT) to C57/BL6 mice. Levels of LSEC markers were up-regulated during the repair phase of MCT-induced hepatotoxicity. The damaged LSECs recovered from the injury by expanding LSECs expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in the peri-central area of MCT-injured livers, while LSECs in the same area of uninjured livers lacked LYVE-1 expression. Bone marrow (BM)-derived cells did not incorporate into the restored LSECs. Tie2 expression was related to LSEC recovery in MCT-injured liver tissue. The resident LSECs neighboring uninjured tissue replace damaged LSECs in MCT-injured livers. Tie2 is involved in LSEC recovery from MCT-induced hepatotoxicity.

Bee Venom in Wound Healing.

Bee venom (BV), also known as api-toxin, is widely used in the treatment of different inflammatory diseases such as rheumatoid arthritis or multiple sclerosis. It is also known that BV can improve the wound healing process. BV plays a crucial role in the modulation of the different phases of wound repair. It possesses anti-inflammatory, antioxidant, antifungal, antiviral, antimicrobial and analgesic properties, all of which have a positive impact on the wound healing process. The mentioned process consists of four phases, i.e., hemostasis, inflammation, proliferation and remodeling. The impaired wound healing process constitutes a significant problem especially in diabetic patients, due to hypoxia state. It had been found that BV accelerated the wound healing in diabetic patients as well as in laboratory animals by impairing the caspase-3, caspase-8 and caspase-9 activity. Moreover, the activity of BV in wound healing is associated with regulating the expression of transforming growth factor (TGF-β1), vascular endothelial growth factor and increased collagen type I. BV stimulates the proliferation and migration of human epidermal keratinocytes and fibroblasts. In combination with polyvinyl alcohol and chitosan, BV significantly accelerates the wound healing process, increasing the hydroxyproline and glutathione and lowering the IL-6 level in wound tissues. The effect of BV on the wounds has been proved by numerous studies, which revealed that BV in the wound healing process brings about a curative effect and could be applied as a new potential treatment for wound repair. However, therapy with bee venom may induce allergic reactions, so it is necessary to assess the existence of the patient’s hypersensitivity to apitoxin before treatment.

The effect of Lucilia sericata larval excretion/secretion (ES) products on cellular responses in wound healing.

Chronic wounds are still regarded as a serious public health concern, which are on the increase mainly due to the changes in life styles and aging of the human population. There are different types of chronic wounds, each of which requires slightly different treatment strategies. Nevertheless, wound bed preparation is included in treatment of all types of chronic wounds and involves tissue debridement, inflammation, and infection control, as well as moisture balance and epithelial edge advancement. Maggot therapy (MT) is a form of biological debridement which involves the application of live medical grade Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae) larvae. Whereas it was initially thought to act mainly through debridement, today MT is known to influence all four overlapping physiological phases of wound repair: homeostasis, inflammation, proliferation, and remodelling/maturing. During MT, medical-grade larvae are applied either freely or enclosed in tea-bag like devices (biobag) inside the wounds, which suggests that larva excretion/secretion (ES) products can facilitate the healing processes directly without the need of direct contact with the larvae. This review summarizes the relevant literature on ES-mediated effects on the cellular responses involved in wound healing.

Pseudomonas aeruginosa virulence proteins pseudolysin and protease IV impede cutaneous wound healing

AbstractThe intricate biological process of cutaneous wound healing is achieved through precise and highly programmed events. Dermal fibroblasts and keratinocytes play a significant role in the process of reepithelialization during wound healing. Pathogenic bacteria such as Pseudomonas aeruginosa (P. aeruginosa) may delay the proliferative phase of wound repair by secreting their proteins leading to delayed or impaired wound healing. We have analyzed three virulent strains of P. aeruginosa isolated from the wound environment which also differed in their ability to produce biofilms. Mass spectrometric analysis of differentially expressed secreted proteins by three virulent strains of P. aeruginosa revealed peptides from pseudolysin and protease IV expressed from lasB and prpL genes. Pseudolysin and protease IV recombinant proteins were tested for their ability to modulate wound healing in several cell types of wound microenvironment in in vitro and in vivo models. Both pseudolysin and protease IV inhibited migration and survival of fibroblasts, keratinocytes, and endothelial cells. In three dimensional spheroid endothelial models and matrigel assays these proteins impeded sprouting and tube formation. In a mouse model of excision wound, pseudolysin and protease IV treatment showed reduced collagen content, inhibited neovascularization and epithelialization, and delayed wound contraction. Furthermore, pseudolysin and protease IV treatment resulted in a significant increase in plasma IL-6 levels when compared to vehicle control and control, suggesting the induction of a state of prolonged inflammation. Taken together, our data indicate pseudolysin and protease IV secreted from biofilm producing and antibiotic resistant P. aeruginosa in wound microenvironment produce both local and systemic effects that is detrimental to the maintenance of tissue homeostasis. Hence, these proteins may serve as potential therapeutic targets toward better clinical management of wounds.

Abstract 15501: Repeated Social Defeat Exaggerates Cacl2-induced Abdominal Aortic Aneurysm Expansion by Suppression of Periaortic Fibrosis in Tissue Repair Phase

Background and Objective: Depression is an independent risk factor of cardiovascular disease and significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We investigated the impact of repeated social defeat (RSD) on AAA formation. Methods and Results: Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same cage without physical contact. After social interaction test to confirm depressive-like behaviors, mice underwent application of 0.5M calcium chloride (CaCl 2 ) on the infrarenal aorta. At one-week, maximum diameter and circumference of external elastic membrane were comparable between the two groups. The number of F-4/80 and MMP-9-positive cells in immunofluorescent images were also comparable. Consistently, fluorescent image taken by IVIS revealed no difference in MMP activity, suggesting that acute inflammatory response was comparable between the two groups. In contrast, at two weeks, maximum diameter and circumference of external elastic membrane were significantly increased in defeated mice ( p &lt; 0.01). Intriguingly, periaortic fibrotic area in aneurysmal portion was markedly decreased in defeated mice (12.5х10 3 μm 2 vs. 3.7х10 3 μm 2 , Control vs. Defeat, p &lt; 0.01). Consistently, accumulation of α-SMA-positive cells in adventitia of aneurysmal portion was much lesser in defeated mice (876 cells/mm 2 vs. 319 cells/mm 2 , Control vs. Defeat, p &lt; 0.05), whereas those in tunica media of non-aneurysmal portion did not show any difference between the two groups. LPS-induced iNOS gene and protein expressions in bone marrow-derived macrophages (BMDMs) were significantly decreased in defeated mice, whereas the TGF-β-induced phosphorylation of Smad2 and ERK1/2 in primary cultured aortic VSMCs were comparable between the two groups. Conclusions: Our findings demonstrate that RSD enhances AAA expansion by suppressing the periaortic fibrosis following inflammatory response, and suggest that decreased NO production from accumulated macrophages contributes to the reduced VSMCs proliferation and collagen deposition.

Abstract 13667: Programming to S1PR1 + Endothelial Cell Population Promotes the Restoration of Vascular Integrity in vivo

Introduction: Increased endothelial permeability and failure to repair is the hallmark of several vascular diseases including acute lung injury (ALI). However, little is known about the intrinsic pathways that activate the endothelial cell (EC) regenerative programs facilitating thereby tissue repair. Studies have invoked a crucial role of sphingosine-1-phosphate (S1P) in resolving endothelial hyperpermeability through activation of the G-protein coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1). Hypothesis: We postulate that S1PR1 + EC serve as an endogenous means to prevent endothelial injury. Methods: Studies were made using EC-S1PR1 null mice and S1PR1-GFP reporter mice to trace the generation and characteristics of S1PR1 + EC by exploiting immuno-histochemical analysis and FACS. RNA-seq analysis was performed to identify the genetic signature of S1PR1 + EC. Combination of genetic and pharmacological strategies were included for mechanistic study. Transplantation of S1PR1 + EC and edema measurement was performed in EC-S1PR1 null mice. Results: We observed in a mouse model of endotoxemia that LPS via generation of S1P induced the programming of S1PR1 lo EC to S1PR1 + EC, comprising 80% of lung EC. Their generation preceded the vascular repair phase and these cells were required for reestablishing the endothelial barrier function. Thus, conditional deletion of S1PR1 in EC spontaneously increased lung vascular permeability. RNA-seq analysis of S1PR1 + EC showed enrichment of genes regulating S1P synthesis and transport, sphingosine kinase 1 (SPHK1) and SPNS2, respectively, as well as transcription factors EGR1 and STAT3. EGR1 and STAT3 were essential for transcribing SPHK1 and SPNS2, respectively to increase S1P concentration that served to amplify S1PR1 + EC transition. Transplantation of S1PR1 + EC into injured lung vasculature of EC-S1PR1 -/- mice restored endothelial integrity. Conclusions: Findings illustrate that generation of a specialized S1PR1 + EC population has the potential to activate key endothelial regenerative program mediating vascular endothelial repair raising the possibility of activating this pathway to restore vascular homeostasis in inflammatory lung injury.

Key criteria, principles and directions for sustainable gold mining

Subject. The article discusses what distinguishes the sustainable development of gold mining in the Russian Federation. Objectives. I herein set up criteria and principles for ensuring the sustainable development of gold mining so as to reach the balance of the economy, environment and social sector throughout places where gold mining enterprises operate. Methods. My theoretical projections are based on the analysis of economic literature and empirical research on resources, environment and sustainable economic development in the existing economic growth areas. Results. I examine the relationship of key criteria and principles for the sustainable development of gold mining. The article aligns directions for the reasonable natural resource use in gold mining, displays opportunities digital technologies give at phases of prospecting, production, processing, trade, maintenance and repair of mining equipment. The article also sets up gold mining investment requirements in terms of further sustainable development. Conclusions and Relevance. The set of gold mining requirements for the Russian Federation will now help flag points for further sustainable development of the gold mining sector as a method to steer and manage the business, ensuring the dynamic growth and balance of related and mutually dependent social, environmental and economic aspects provided they could be regularly and constantly updated. The set of requirements will bring the gold mining sector to the forefront of the Russian industrial production. If a 50 tonnes limit is raised or lifted, the sector will more easily apply for foreign investment and increase the finance of geological prospecting, since the 50 tonnes limit restricted foreign investment in federal subsoil areas.

Interleukin-6 (IL-6) deficiency enhances intramembranous osteogenesis following stress fracture in mice.

Interleukin-6 (IL-6) is highly upregulated in response to skeletal injury, suggesting it plays a role in the inflammatory phase of fracture repair. However, the impact of IL-6 on successful repair remains incompletely defined. Therefore, we investigated the role of IL-6 in two models of fracture repair (full fracture and stress fracture) using 12-week old IL-6 global knockout mice (IL-6 KO) and wild type (WT) littermate controls. Callus morphology and mineral density 14days after full femur fracture did not differ between IL-6 knockout mice and controls. In contrast, IL-6 KO mice had an enhanced bone response 7days after ulnar stress fracture compared to WT, with increased total callus volume (p=0.020) and callus bone volume (p=0.045). IL-6 KO did not alter the recruitment of immune cells (Gr-1 or F4/80 positive) to the stress fracture callus. IL-6 KO also did not alter the number of osteoclasts in the stress fracture callus. Using RNA-seq, we identified differentially expressed genes in stress fracture vs. contralateral control ulnae, and observed that IL-6 KO resulted in only modest alterations to the gene expression response to stress fracture (SFx). Wnt1 was more highly upregulated in IL-6 KO SFx callus at both day 1 (fold change 12.5 in KO vs. 5.7 in WT) and day 3 (fold change 4.7 in KO vs. 1.9 in WT). Finally, using tibial compression to induce bone formation without bone injury, we found that IL-6 KO directly impacted osteoblast function, increasing the propensity for woven bone formation. In summary, we report that IL-6 knockout enhanced formation of callus and bone following stress fracture injury, likely through direct action on the osteoblast's ability to produce woven bone. This suggests a novel role of IL-6 as a suppressor of intramembranous bone formation.

Morphogenesis and histogenesis during the arm regeneration in a basket star Astrocladus dofleini (Euryalida, Ophiuroidea, Echinodermata).

Basket stars, that is, Ophiuroidea in Echinodermata, exhibit distinctive morphological characteristics with their complicatedly branched arms that can regenerate immediately after mutilation. Although, in brittle stars, that is, ophiuroids with nonbranched arms, the arm regeneration process following accidental trauma or autotomy have been morphologically and histologically observed in several species, few studies have so far been carried out on the regeneration of branched arms in basket stars. In this study, the developmental and morphological features of arm regeneration in Astrocladus dofleini (Gorgonocephalidae, Euryalida, Euryophiurida), one of the most common basket star species in Japanese waters, was anatomically and histologically investigated. Results clearly showed the following phases during the arm regeneration: (a) repair phase, (b) early regenerative phase, (c) intermediate regenerative phase, (d) advanced regenerative Phase I, and (e) advanced regenerative Phase II. The morphogenetic process during the arm regeneration in the basket star showed similar patterns to those of nonbranched arms observed in other ophiuroids. However, differences were also seen between the two ophiuroid types, that is, there were some developmental features specific to the basket star. In the early regenerative phase, branching of coelomic cavities was observed prior to the formation of other tissues, probably inducing the later morphogenesis of branched arms. In addition, hard skeletal ossicles form rapidly at the advanced regenerative Phase II. These developmental features may have led the evolution of bizarre morphologies seen in basket stars, probably contributing to the adaptation to shallow waters from deep-sea habitats.

Repeated social defeat exaggerates CaCl2-induced abdominal aortic aneurysm expansion by eliminating periaortic fibrosis in tissue repair phase: possible involvement of specific subtypes of macrophages

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD) and significantly associated with the prevalence of abdominal aortic aneurysm (AAA). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviors in apoE−/− mice and exaggerates atherosclerosis development by enhancing leukocyte activation. Here, we investigated the impact of RSD on AAA formation. Methods and results Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (28 of 48) and control mice (31 of 36) underwent application of 0.5 M calcium chloride (CaCl2) on the infrarenal aorta to induce AAA. At 1 week after application, maximum diameter and circumference of external elastic membrane were comparable between the two groups. The number of F-4/80, MMP-9, and TNF-α-positive cells in immunofluorescent images were also comparable. Further, in vitro bone marrow derived macrophages stimulation by LPS did not show any difference in mRNA expression levels of inflammatory cytokines, suggesting no discernable difference in acute inflammatory response between the two groups. In contrast, at 2 weeks after application, at the time point when MMP-9 and TNF-α-positive cells were scarcely observed, maximum diameter and circumference of external elastic membrane were significantly increased in defeated mice (0.72 mm vs. 0.90 mm, 1.59 mm vs. 2.00 mm, respectively, Control vs. Defeat, p&amp;lt;0.01). Intriguingly, periaortic fibrotic area in aneurysmal portion was markedly decreased in defeated mice (12.5×103 μm2 vs. 3.7×103 μm2, Control vs. Defeat, p&amp;lt;0.01). Consistently, accumulation of α-SMA-positive cells in adventitia of aneurysmal portion was much less in defeated mice than control mice (876 cells/mm2 vs. 319 cells/mm2, Control vs. Defeat, p&amp;lt;0.05), whereas those in tunica media of non-aneurysmal portion did not show any difference between the two groups. We next focused on the segregated nucleus-containing atypical monocyte (SatM), specific subtypes of monocytes/macrophages that are involved in fibrosis in injured tissues during the healing phase. We could observe SatM fraction in AAA tissue of control mice using flow cytometry. We also found that mRNA expression level of C/EBPβ, an essential regulator for SatM differentiation, was markedly decreased by 76% in BM cells of defeated mice compared with control mice (p&amp;lt;0.05). Conclusions Our findings demonstrate for the first time that RSD enhances AAA expansion by eliminating periaortic fibrosis in tissue repair phase, suggesting that the impaired resolution of acute inflammation after CaCl2 application contributes, at least in part, to the augmented expansion of AAA in defeated mice. Funding Acknowledgement Type of funding source: None

YAP activity protects against endotoxemic acutelung injury by activating multiple mechanisms.

The roles of the Hippo‑Yes‑associated protein (YAP) pathway in lung injury and repair remain elusive. The present study examined the effects of systemic inhibition or stimulation of YAP activity on lung injury, repair and inflammation in a mouse model of lipopolysaccharide(LPS)‑induced lung injury. Mice were treated with or without YAP inhibitor, verteporfin, or with or without YAP stimulator, XMU‑MP‑1, and intraperitoneally injected with LPS (7.5mg/kg). Lung injury and repair were evaluated by histological analysis and by testing for markers of lung injury. Lung inflammation was assessed by measuring tissue levels of inflammatory mediators. Lung injury was associated with a decreased, whereas lung repair was associated with an increased YAP activity evidenced by nuclear translocation. Lung injury was associated with a high level of lung inflammation and epithelial adherens junction disassembly, but not with cell proliferation or epithelial cell regeneration. The injury phase was defined as 0‑48h post‑LPS injection, and the 48‑168h time period was considered the repair phase. Inhibition of YAP activity at the injury phase, using verteporfin, exacerbated, whereas its stimulation, using XMU‑MP‑1, alleviated lung injury, lung inflammation and epithelial adherens junction disassembly. Inhibition or stimulation of YAP activity at the injury phase had no effects on cell proliferation or epithelial regeneration. By contrast, lung repair was associated with inflammation resolution, increased cell proliferation, epithelial regeneration and reassembly of epithelial adherens junctions. Inhibition of YAP activity at the repair phase delayed inflammation resolution, impeded lung recovery, inhibited cell proliferation and epithelial regeneration, and inhibited epithelial adherens junction reassembly. Stimulation of YAP activity at the repair phase reversed all these processes. The results of the current study demonstrated that the Hippo‑YAP activity serves a protective role against endotoxemic lung injury. The Hippo‑YAP activity alleviated lung inflammation and injury at the injury phase and promoted inflammation resolution and lung repair at the repair phase.

Enhancement of nutritional soy protein and peptide supplementation on skin repair in rats

• Protein supplements play a vital role during the inflammatory phase of skin repair. • Soy peptide performed better on fibroblast proliferation than the soy protein. • Soy peptide performed better on collagen synthesis than the soy protein. • Soy peptide showed a better performance of wound tissue repair. The aim of this study was to assess the effect of soy protein and soy peptide on skin repair process in male Wistar rats. Soy protein and soy peptide were administered orally by gavage to the rats post-operatively for 16 days. The supplementation of soy protein and soy peptide resulted in improvement of leucocytes production, decrease in serum inflammation index (IL-1β, IL-6, TNF-α) as well as increase of plasma protein concentration. Additionally, the excessive production of inflammatory factors in peptide group were significantly suppressed compared with the protein group at day 4, which indicated that soy peptide had a better impact on promoting the process of skin repair. Histopathological analysis indicated that peptide group performed better on fibroblast proliferation and collagen synthesis than the protein group, and eventually manifests as more collagen deposition and a better skin repair rate. In conclusion, soy peptide displayed the potential to promote skin regeneration.

ÇELİK FİBERLİ ASFALT BETONU KARIŞIMLARIN MARSHALL STABİLİTESİ VE İNDİREKT ÇEKME MUKAVEMETİNİN İNCELENMESİ

One of the conductive fiber types used in the mixtures in order to provide the desired performance by reducing the deterioration of asphalt concrete coatings during the project is steel fibers. In recent years, studies on the use of steel fiber to provide self-healing property by heating the layers during the repair phase have attracted attention. In order to evaluate the economy of steel fiber layers, it is necessary to know the mechanical properties of these layers before they come into repair. In this study, the abrasion made Marshall design to binder mixtures, according to the design result, at the rate of 0.1%, 0.15% and 0.2% of the total weight of aggregate and bitumen, a steel fiber of 10 mm length and 1 mm diameter was added to the mixtures. After the first Marshall stability, conditioned indirect tensile strength tests on the mixtures, the same tests were applied again without removing the samples from the device. As a result, the highest Marshall stability and conditioned indirect tensile strength were obtained in binder mixes with 0.10% steel fiber compared to the unadulterated, while in the additive wear mixture tests, close to the additive-free or lower results were obtained.

Drill Hole Models to Investigate Bone Repair.

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

A dual-phase scaffold produced by rotary jet spinning and electrospinning for tendon tissue engineering

Tendon is a highly hierarchical and oriented tissue that provides high mechanical strength. Tendon injuries lead to loss of function, disability, and a decrease in quality of life. The limited healing capacity of tendon tissue leads to scar tissue formation, which can affect mechanical strength and cause a re-tear. Tissue engineering can be the solution to achieving complete and proper healing of tendon. The developed constructs should be mechanically strong while maintaining a suitable environment for cell proliferation. In this study, a dual-phase fibrous scaffold was produced by combining fibrous mats produced by rotary jet spinning (RJS) and wet electrospinning (WES), with the intent of improving the healing capacity of the construct. Dual-phase scaffolds were formed from aligned poly(ϵ-caprolactone) (PCL) fibers (Shell) produced by RJS and randomly oriented PCL or PCL/gelatin fibers (Core) produced by WES systems. The scaffolds mimicked i) the repair phase of tendon healing, in which randomly-oriented collagen type III is deposited by randomly-oriented WES fibers and ii) the remodeling stage, in which aligned collagen type I fibers are deposited by aligned RJS fibers. In vitro studies showed that the presence of randomly-oriented core fibers inside the aligned PCL fiber shell of the dual-phase scaffold increased the initial attachment and viability of cells. Scanning electron microscopy and confocal microscopy analysis showed that the presence of aligned RJS fibers supported the elongation of cells through aligned fibers which improves tendon tissue healing by guiding oriented cell proliferation and extracellular matrix deposition. Tenogenic differentiation of human adipose-derived mesenchymal stem cells on scaffolds was studied when supplemented with growth differentiation factor 5 (GDF-5). GDF-5 treatment improved the viability, collagen type III deposition and scaffold penetration of human adipose derived stem cells. The developed FSPCL/ESPCL-Gel 3:1 scaffold (FS = centrifugal force spinning/RJS, ES = wet electrospinning, Gel = gelatin) sustained high mechanical strength, and improved cell viability and orientation while supporting tenogenic differentiation.

Prediction of the crosstalk regulation model between the abscisic acid (ABA) signaling and peroxisome abundance during drought stress in wheat (Triticum aestivum L.)

Phenotyping plant responses under the progressive phases of drought stress are complicated by tangled signaling pathways. To explore one of these complex signaling networks we investigated the relationship between the abscisic acid (ABA) hormone signaling pathway, plant peroxisome abundance as a ROS scavenger during the response and repair phases of drought. Photosynthetic activity, morphological traits, and plant yield were measured in response to imposed drought stress, and this data was used along with published transcriptomic data for wheat (Triticum aestivum) to identify regulatory genes associated with distinct drought phases, as well as to predict novel protein networks associated with drought response. We found that greater phenotypic variation occurred during the repair phase than the response phase of drought, ABA content positively correlated with peroxisome abundance at the repair phase but negatively correlated with the other traits under both drought phases, and drought upregulated TaABA4, TaPEX11.A, TaDRP3B, TaDRP5A, and TaFIS1A genes, while downregulating TaLHB1B2, TaLHCA1, and TaPsbR genes. We also found that TaFIS1A, is a hub protein for the peroxisome biogenesis protein network, shared the same cluster with ABA biosynthesis proteins and TaLHCA1 protein. This study provides new evidence for a connection between ABA signaling, peroxisome proliferation, and drought regulation.

Concerted actions by MMPs, ADAMTS and serine proteases during remodeling of the cartilage callus into bone during osseointegration of hip implants

IntroductionAlthough the number of patients undergoing total hip arthroplasty is constantly on the rise, we only have limited knowledge of the molecular mechanisms necessary for successful osseointegration of implants or the reasons why some fail. Understanding the spatiotemporal characteristics of signaling pathways involved in bone healing of implants is therefore of particular importance for our ability to identify factors causing implants to fail. The current study investigated the role of three families of proteases, i.e. MMPs (matrix metalloproteinases), ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and serine proteases, as well as their endogenous inhibitors during osseointegration of hip implants that have endured two decades of use without clinical or radiological signs of loosening. Materials and methodsTwenty-four patients that had undergone primary THA due to one-sided osteoarthritis (OA) were monitored during 18 years (Y) with repeated measurements of plasma biomarkers, clinical variables and radiographs. All implants were clinically and radiographically well-fixed throughout the follow-up. Eighty-one healthy donors divided in three gender and age-matched groups and twenty OA patients awaiting THA, served as controls. Plasma was analyzed for MMP-1, -2, -3, -8, -9, -10, -13, -14, tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, ADAMTS4, ADAMTS5, the serine proteases neutrophil elastase (NE), proteinase 3 (PR3) and their endogenous inhibitors, secretory leucocyte proteinase inhibitor (SLPI), trappin-2/elafin and serpina1 (α-1 antitrypsin). Cartilage turnover was monitored using two markers of cartilage synthesis, type II procollagen and PIICP (procollagen II C-terminal propeptide), and two markers of cartilage degradation, CTX-II (C-terminal telopeptide fragments of type II collagen) and split products of aggrecan (G1-IGD-G2). ResultsMMP-1, MMP-9, ADAMTS4, NE and PR3 were above healthy in presurgery OA patients but returned to the level of healthy within 6 weeks (W) after surgery. MMPs and serine proteases were counter-regulated during this phase by TIMP-1, SLPI and trappin-2/elafin. Type II procollagen, PIICP and CTX-II increased to a peak 6 W after surgery with a gradual return to the level of controls within weeks. Significant increases by MMP-8, MMP-9, ADAMTS4, ADAMTS5, NE, PR3 and the protease inhibitors, TIMP-3 and serpina1, were seen 5 Y after hip arthroplasty paralleled by a sharp increase in the levels of the cartilage degradation markers, CTX-II and G1-IGD-G2. All the above mediators were normalized before 18 Y, except MMP-1 and MMP-9 that remained above healthy at 18 Y. MMP-14 increased immediately after surgery and remained elevated until 5 Y postsurgery before returning to the level of controls at 7 Y. ConclusionNotwithstanding temporal differences, the molecular processes of bone repair in arthroplasty patients show great spatial similarities with the classical phases of fracture repair as previously shown in animal models. Cartilagenous callus, produced and remodeled early after hip arthroplasty, is replaced with bone 5 Y to7 Y after surgery by the concerted actions of MMP-8, MMP-9, ADAMTS4, ADAMTS5, NE and PR3, thus suggesting that a complex regulatory cross-talk may exist between different families of proteases during this transitional phase of cartilage degradation. Regulation and fine-tuning of cartilage remodeling by MMPs and ADAMTS is controlled by TIMP-3 whereas serine proteases are regulated by serpina1. Increased MMP-1 and MMP-9 beyond 10Y post-THA support a role during coupled bone remodeling.

DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology-all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.