scholarly journals Defining the activation profile and fate trajectory of adult Scleraxis-lineage cells during tendon healing by combining lineage tracing and spatial transcriptomics

2021 ◽  
Author(s):  
Jessica E Ackerman ◽  
Katherine T Best ◽  
Samantha N Muscat ◽  
Chia-Lung Wu ◽  
Alayna E Loiselle
Keyword(s):  
Time Course ◽  
Molecular Mechanisms ◽  
Healing Process ◽  
Tendon Repair ◽  
Tendon Healing ◽  
Cell Types ◽  
Healing Time ◽  
Lineage Tracing ◽  
Repair Site ◽  
Temporal And Spatial

The tendon healing process is regulated by the coordinated interaction of multiple cell types and molecular processes. However, these processes are not well-defined leading to a paucity of therapeutic approaches to enhance tendon healing. Scleraxis-lineage (ScxLin) cells are the major cellular component of adult tendon and make time-dependent contributions to the healing process. Prior work from our lab and others suggests heterogeneity within the broader ScxLin population over the course of tendon healing; therefore delineating the temporal and spatial contributions of these cells is critical to understanding and improving the healing process. In the present study we utilize lineage tracing of the adult ScxLin population to determine whether these cells undergo cellular activation and subsequent myofibroblast differentiation, which is associated with both proper healing and fibrotic progression in many tissues. We show that adult ScxLin cells undergo transient activation in the organized cellular bridge at the tendon repair site, contribute to the formation of an organized neo-tendon, and contribute to a persistent myofibroblast population in the native tendon stubs. The mechanisms dictating this highly specialized spatial response are unknown. We therefore utilized spatial transcriptomics to better define the spatio-molecular program of tendon healing. Integrated transcriptomic analyses across the healing time-course identifies five distinct molecular regions, including key interactions between the inflammatory bridging tissue and highly reactive tendon tissue at the repair site, with adult ScxLin cells being a central player in the transition from native tendon to reactive, remodeling tendon. Collectively, these data provide important insights into both the role of adult ScxLin cells during healing as well as the molecular mechanisms that underpin and coordinate the temporal and spatial healing phenotype, which can be leveraged to enhance the healing process.

scholarly journals The Thioredoxin Family Proteins: Histopathological Time Course Study in the Asphyctic Male Rat Brain

2020 ◽  
Vol 26 (S1) ◽  
pp. 183-184
Author(s):  
Ma. Laura Aón Bertolino ◽  
Christopher Horst Lillig ◽  
Capani Francisco
Keyword(s):  
Structural Damage ◽  
Cellular Localization ◽  
Time Course ◽  
Redox Regulation ◽  
Molecular Mechanisms ◽  
Neuroprotective Effect ◽  
Cell Types ◽  
Initial Time ◽  
Male Rat ◽  
Morphological Evidence

Summary:Thioredoxin Family of proteins as Thioredoxin (Trxs), Glutaredoxins (Grxs) and Peroxiredoxins (Prxs) are one of the most important agents in the defense of oxidative  stress  and  redox regulation. Perinatal asphyxia (AP) a disorder generated at the expense of the deficit of oxygen associated or not to ischemia, affects 5 to 10 of every 1,000 live births in developing country and is a serious health problem worldwide. Alterations in antioxidant protection systems are involved in the pathogenesis of hypoxic-ischemic insult and neuronal death. For these reasons it is proposed that the  AP can cause changes in the distribution and expression of antioxidant proteins and enhance their deleterious or neuroprotective effects on the CNS. Methods: to determine the implication of the  proteins role induced in the hypoxic brain injury, an animal model in vivo of PA was used in this    work. In the first instance, the identification of the distribution of Trxs family proteins Trx1, Trx2, TrxR1, TrxR2, Txnip, Grx1, Grx2, Grx3, Grx5, γ-GCS, Prx1, Prx2, Prx3, Prx4, Prx5 and Prx6 was performed by immunohistochemistry on areas most sensitive to a  hypoxia-ischemia  insult:  cerebellum, striatum, hippocampus, spinal cord, sustantia nigra, cortex and retina. Previous studies suggest that these proteins have an extensive and characteristic distribution in various cell types and regions of the CNS, although we observed significant differences in labeling intensity and distribution with conventional and fluorescence optical microscopy. After determining the cellular localization of Trxs, their behavior was studied by during a hypoxic- ischemic event by setting a time course at different times (2, 4, 6, 12, 24 and 72 hours post AP). Results: Trx1, Trx2, Grx1 and Grx2 proteins constitutes the main oxidoreductases in the cytosol and mitochondria, both in physiological and pathological conditions. Thus, for the short asphyxia times in which they were studied, Trx1 was detected with increased expression at 2 hours and up to 4 hours post-AP, arguing a probable neuroprotective effect, although not enough, if taken  into account tissue liability and high levels  of  free radicals detected at initial hours and the presence of structural damage at 7 days post-AP. Trx2 shows its increase at 6 and 12 hours and Grx1 at 24 hours. For Grx2, values elicited with the ELISA technique in the initial time curve were not representative in their expression in the hippocampus, contrary to the findings in the distribution in the striatum, where Grx2 was observed to be increased. Although at 7 hours post-AP levels begin to decline, these  redoxins appears to be essential  in the  initial response to injury. Conclusions: by demonstrating of the presence of these enzymes in different cell types and specific regions of the brain in rats subjected to AP provided us with important morphological evidence base to study later some of the molecular mechanisms involved in the pathogenesis of cerebral ischemia that will help to generate new therapeutic tools to mitigate a disease whose neurological complications have no definitive  solution.

Application of a Purified Protein From Natural Latex and the Influence of Suture Type on Achilles Tendon Repair in Rats

2019 ◽  
Vol 47 (4) ◽  
pp. 901-914 ◽  
Author(s):  
Diego Pulzatto Cury ◽  
Bárbara Tavares Schäfer ◽  
Sonia Regina Yokomizo de Almeida ◽  
Marta Maria da Silva Righetti ◽  
Ii-sei Watanabe
Keyword(s):  
Healing Process ◽  
Tendon Healing ◽  
Healing Time ◽  
Suture Technique ◽  
Collagen Fibrils ◽  
Control Group ◽  
Slight Reduction ◽  
Type I ◽  
Sprague Dawley ◽  
Normal Tendon

Background: The prolonged tendon-healing process, the high costs associated with treatment, the increase in the number of injuries over the past decades, and the lack of consensus on the optimal treatment of tendon injuries are a global problem. Restoring the normal tendon anatomy and decreasing the healing time are key factors for treatment advancement. Hypothesis: Application of a purified protein from natural latex (PPNL) accelerates the healing process, increasing collagen synthesis and decreasing metalloproteinases. PPNL associated with a simpler suture technique should decrease the healing time. Study Design: Controlled laboratory study. Methods: Injury, surgery, and treatment with PPNL were conducted with male Sprague-Dawley rats. Two suture techniques were used: U-suture, a simpler and lesser traumatic technique, and Kessler-Tajima, to avoid strangulation of the microcirculation. Achilles tendons were completely sectioned, and 100 µL of 0.1% PPNL was applied on the tendon during surgery. Tendon morphology, distribution, and quantity of collagen types I and III, as well as expression of TIMP-1, TIMP-2, MMP-2, and MMP-9 and ultrastructural aspects of cells and collagen fibrils, were assessed after 2 and 4 weeks. Results: PPNL treatment improved collagen type I synthesis and reduced MMP-2 expression. All groups showed a 6.8-times increase in tendon weight as compared with the control group after 2 weeks and a 5.2-times increase after 4 weeks. All groups showed an increase in diameter after 4 weeks, except for the ones treated with PPNL, which showed a slight reduction in diameter. The peak of concentration of collagen fibrils with a 80-nm diameter was 27.79% in the control group; all other experimental groups presented fibrils between 50 and 60 nm. However, the best results were observed with Kessler-Tajima suture associated with PPNL. Conclusion/Clinical Relevance: There are no known medicines or substances capable of aiding the tendon healing process besides surgery. The discovery of a substance able to improve this process and decrease its duration represents an important advancement in orthopaedic medicine.

scholarly journals Basic Research on Tendon Repair: Strategies, Evaluation, and Development

2021 ◽  
Vol 8 ◽  
Author(s):  
Zhi Jie Li ◽  
Qian Qian Yang ◽  
You Lang Zhou
Keyword(s):  
Wound Healing ◽  
Platelet Rich Plasma ◽  
Healing Process ◽  
Tendon Repair ◽  
Continuous Optimization ◽  
Tendon Healing ◽  
Basic Research ◽  
Clinical Problem ◽  
Tendon Injury ◽  
Wound Healing Process

Tendon is a fibro-elastic structure that links muscle and bone. Tendon injury can be divided into two types, chronic and acute. Each type of injury or degeneration can cause substantial pain and the loss of tendon function. The natural healing process of tendon injury is complex. According to the anatomical position of tendon tissue, the clinical results are different. The wound healing process includes three overlapping stages: wound healing, proliferation and tissue remodeling. Besides, the healing tendon also faces a high re-tear rate. Faced with the above difficulties, management of tendon injuries remains a clinical problem and needs to be solved urgently. In recent years, there are many new directions and advances in tendon healing. This review introduces tendon injury and sums up the development of tendon healing in recent years, including gene therapy, stem cell therapy, Platelet-rich plasma (PRP) therapy, growth factor and drug therapy and tissue engineering. Although most of these therapies have not yet developed to mature clinical application stage, with the repeated verification by researchers and continuous optimization of curative effect, that day will not be too far away.

scholarly journals How a cell decides its own fate: a single-cell view of molecular mechanisms and dynamics of cell-type specification

2021 ◽  
Author(s):  
Maria Mircea ◽  
Stefan Semrau
Keyword(s):  
Single Cell ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Lineage Tracing ◽  
Molecular Profile ◽  
Cell Type ◽  
Molecular Changes ◽  
Starting Point ◽  
A Cell ◽  
Type Specification

On its path from a fertilized egg to one of the many cell types in a multicellular organism, a cell turns the blank canvas of its early embryonic state into a molecular profile fine-tuned to achieve a vital organismal function. This remarkable transformation emerges from the interplay between dynamically changing external signals, the cell's internal, variable state, and tremendously complex molecular machinery; we are only beginning to understand. Recently developed single-cell omics techniques have started to provide an unprecedented, comprehensive view of the molecular changes during cell-type specification and promise to reveal the underlying gene regulatory mechanism. The exponentially increasing amount of quantitative molecular data being created at the moment is slated to inform predictive, mathematical models. Such models can suggest novel ways to manipulate cell types experimentally, which has important biomedical applications. This review is meant to give the reader a starting point to participate in this exciting phase of molecular developmental biology. We first introduce some of the principal molecular players involved in cell-type specification and discuss the important organizing ability of biomolecular condensates, which has been discovered recently. We then review some of the most important single-cell omics methods and relevant findings they produced. We devote special attention to the dynamics of the molecular changes and discuss methods to measure them, most importantly lineage tracing. Finally, we introduce a conceptual framework that connects all molecular agents in a mathematical model and helps us make sense of the experimental data.

scholarly journals In vitro wound healing assays – state of the art

2016 ◽  
Vol 17 (1-2) ◽  
Author(s):  
Anne Stamm ◽  
Kerstin Reimers ◽  
Sarah Strauß ◽  
Peter Vogt ◽  
Thomas Scheper ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Healing Process ◽  
Cell Monolayer ◽  
3D Culture ◽  
Cell Types ◽  
In Vitro Assays ◽  
Complex Wound

AbstractWound healing is essential for the restoration of the barrier function of the skin. During this process, cells at the wound edges proliferate and migrate, leading to re-epithelialization of the wound surface. Wound healing assays are used to study the molecular mechanisms of wound repair, as well as in the investigation of potential therapeutics and treatments for improved healing. Numerous models of wound healing have been developed in recent years. In this review, we focus on in vitro assays, as they allow a fast, cost-efficient and ethical alternative to animal models. This paper gives a general overview of 2-dimensional (2D) cell monolayer assays by providing a description of injury methods, as well as an evaluation of each assay’s strengths and limitations. We include a section reviewing assays performed in 3-dimensional (3D) culture, which employ bioengineered skin models to capture complex wound healing mechanics like cell-matrix interactions and the interplay of different cell types in the healing process. Finally, we discuss in detail available software tools and algorithms for data analysis.

scholarly journals Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse

2021 ◽  
Author(s):  
Jacob G. Kallenbach ◽  
Margaret A. T. Freeberg ◽  
David Abplanalp ◽  
Jacquelyn A. Myers ◽  
John M. Ashton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Cellular Proliferation ◽  
Tendon Repair ◽  
Tendon Healing ◽  
Biomechanical Properties ◽  
Transcriptional Analysis ◽  
Flexor Tendon Repair ◽  
Cytokine Analysis ◽  
Mrl Mouse

AbstractTo better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth’s Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. However, only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins (SASP) in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.

Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage

Development ◽  
1999 ◽  
Vol 126 (22) ◽  
pp. 5041-5049 ◽  
Author(s):  
G. Cheng ◽  
W.H. Litchenberg ◽  
G.J. Cole ◽  
T. Mikawa ◽  
R.P. Thompson ◽  
...  
Keyword(s):  
Time Course ◽  
Molecular Mechanisms ◽  
Selection Process ◽  
Conduction System ◽  
Lineage Tracing ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Cardiac Tissues ◽  
Cardiac Pacemaking ◽  
Peripheral Cells

The cardiac pacemaking and conduction system sets and maintains the rhythmic pumping action of the heart. Previously, we have shown that peripheral cells of the conduction network in chick (periarterial Purkinje fibers) are selected within a cardiomyogenic lineage and that this recruitment occurs as a result of paracrine cues from coronary arteries. At present, the cellular derivation of other elements of this specialized system (e.g. the nodes and bundles of the central conduction system) are controversial, with some proposing that the evidence supports a neurogenic and others a myogenic origin for these tissues. While such ontological questions remain, it is unlikely that progress can be made on the molecular mechanisms governing patterning and induction of the central conduction system. Here, we have undertaken lineage-tracing strategies based on the distinct properties of replication-incompetent adenoviral and retroviral lacZ-expressing constructs. Using these complementary approaches, it is shown that cells constituting both peripheral and central conduction tissues originate from cardiomyogenic progenitors present in the looped, tubular heart with no detectable contribution by migratory neuroectoderm-derived populations. Moreover, clonal analyses of retrovirally infected cells incorporated within any part of the conduction system suggest that such cells share closer lineage relationships with nearby contractive myocytes than with other, more distal elements of the conduction system. Differentiation birthdating by label dilution using [(3)H]thymidine also demonstrates the occurrence of ongoing myocyte conscription to conductive specialization and provides a time course for this active and localized selection process in different parts of the system. Together, these data suggest that the cardiac conduction system does not develop by outgrowth from a prespecified pool of ‘primary’ myogenic progenitors. Rather, its assembly and elaboration occur via processes that include progressive and localized recruitment of multipotent cardiomyogenic cells to the developing network of specialized cardiac tissues.

The Effect of Myostatin (GDF-8) on Proliferation and Tenocyte Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells

2017 ◽  
Vol 22 (02) ◽  
pp. 200-207 ◽  
Author(s):  
Wei Le ◽  
Jeffrey Yao
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Tendon Repair ◽  
Tendon Healing ◽  
Sprague Dawley ◽  
Repair Site ◽  
Migration Assay ◽  
Immunohistochemistry Staining ◽  
Rat Bone

Background: The future in flexor tendon surgery involves tissue engineering approaches directed toward increasing early repair strength to accelerate tendon healing and to allow for earlier onset of rehabilitation. Previous work has shown that pluripotential mesenchymal stem cells may be successfully delivered to a tendon repair site using a suture carrier. The current work describes the use of Myostatin (GDF-8) to help guide these delivered pluripotential stem cells to differentiate down a tenocyte lineage to potentially maximize the reparative effects of these cells at the tendon repair site.Methods: Primary rat bone marrow mesenchymal stem cells isolated from the long bones of male Sprague-Dawley rats were treated with 500 ng/ml myostatin for 24 h, 48 h, and 72 h. Collagen 1 A, scleraxis (Scx), and tenomodulin (Tnmd) expression, indicative of tenogenesis, was analyzed using real time PCR and immunohistochemistry staining. A migration assay was performed to assess the functional activity of BMSCs after they were treated with myostatin.Results: Compared to the control cells (without treatment), the cells treated with 500 ng/ml myostatin for 72 h exhibited higher expression of Col 1A, Scx, and Tnmd. The mRNA expression of Col1A, Scx, Tnmd increased 15.3, 13 and 7 times respectively. Immunohistochemistry staining showed Scx and Tnmd were expressed in the cellular cytoplasm. In response to myostatin, the cells also showed a tendency to proliferate and migrate more than the control cells.Conclusions: Myostatin (GDF-8) has the ability to increase rat bone marrow mesenchymal stem cell growth and differentiation toward a tenocyte lineage. This information could be useful for future studies regarding tendon repair.

scholarly journals Short-Duration RAGE Antagonism Transiently Disrupts Tendon Homeostasis and does not Alter Diabetic Tendon Healing

2021 ◽  
Author(s):  
Anne EC Nichols ◽  
Samantha N Muscat ◽  
Alayna E Loiselle
Keyword(s):  
Advanced Glycation Endproducts ◽  
Healing Process ◽  
Tendon Repair ◽  
Tendon Healing ◽  
Type Ii Diabetes Mellitus ◽  
Tendon Injury ◽  
Acute Injury ◽  
Molecular Profile ◽  
Diet Induced Obesity ◽  
Increased Risk

Obesity and type II Diabetes Mellitus (T2DM) have substantial pathological effects on tendon homeostasis, including loss of collagen organization and increased risk of tendon rupture. Moreover, following rupture or acute injury, the healing process is impaired by T2DM. We have previously demonstrating that restoring normal metabolic function in a murine model of obesity/ T2DM is insufficient to blunt or reverse the progression of diabetic tendinopathy, indicating the need for identification of novel therapeutic approaches to both maintain tendon homeostasis, and to improve the healing process. RAGE, the Receptor for Advanced Glycation Endproducts has been implicated as a key driver of several diabetic pathologies. We have demonstrated that pharmacological antagonism of RAGE is sufficient to partially improve tendon healing in non-diabetic animals. Therefore, in the current study we tested the efficacy of blunted RAGE signaling, via treatment with a RAGE Antagonist Peptide (RAP), to improve tendon healing in the context of T2DM. While our study did not find a beneficial effect of short-term RAP treatment on the healing process of T2DM mice, we did identify several important challenges brought about by this model of diet-induced obesity and T2DM. Both high fat (HFD) and low fat diet (LFD) feeding shifted the temporal molecular profile of healing compared to standard laboratory chow fed mice. Moreover, RAP treatment resulted in a transient disruption in homeostasis in the contralateral control tendons of both HFD and LFD mice, and this was due to a potential interaction with the systemic response to tendon injury as this response was not observed in HFD and LFD fed mice that did not undergo tendon repair surgery. Collectively, these data highlight the complications associated with models of diet induced obesity, and the lean control diets that should be considered in future studies.

Wound With Diabetes: Present Scenario And Future

2020 ◽  
Vol 16 ◽  
Author(s):  
Kuldeep B. Pawar ◽  
Shivani Desai ◽  
Ramesh R. Bhonde ◽  
Ritesh P. Bhole ◽  
Atul A. Deshmukh
Keyword(s):  
Wound Healing ◽  
Blood Flow ◽  
Healing Process ◽  
Endocrine System ◽  
Healing Time ◽  
Special Focus ◽  
Diabetic Wound ◽  
Management Options ◽  
And Migration ◽  
Proliferation And Migration

: Diabetes is a chronic metabolic disorder of endocrine system characterized by increase in blood glucose level. Several factors such as pancreatic damage, oxidative stress, infection, genetic factor, obesity, liver dysfunction play a vital role in pathogenesis of diabetes which further lead to serious diabetic complications. Diabetic wound is one such complication where the wound formation occurs, especially due to pressure and its healing process is disrupted due to factors such as hyperglycemia, neuropathy, nephropathy, peripheral vascular disease, reduction of blood flow, atherosclerosis, impaired fibroblast. Process of wound healing is delayed due to different abnormalities like alteration in nitric oxide level, increase in aldose reductase, sorbitol and fructose. Therefore, diabetic wound requires more time to heal as compare to normal wound. Healing time is delayed in diabetic wound due to many factors such as stress, decreased oxygenation supply, infection, decreased blood flow, impaired proliferation and migration rate, impaired growth factor production, impaired keratinocytes proliferation and migration, and altered vascular endothelial mediators. The current treatment for diabetic wound includes wound patches, oxygenation therapy, hydrogel patches, gene therapy, laser therapy, and stem cell therapy. Medications with phytoconstituents is also one way to manage diabetic wound, but it is not more effective for quick healing. The objective of this review is to understand the potential of various management options which are available for diabetic wound, with a special focus on biological cells.

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