Regenerative Growth Research Articles

Cytotoxicity and Gene Expression Changes of a Novel Homeopathic Antiseptic Oral Rinse in Comparison to Chlorhexidine in Gingival Fibroblasts.

Most available antiseptic solutions available today have strong antibacterial effects, however most also possess major cytotoxic effects on human gingival tissues. The VEGA Oral Care Recovery Kit (StellaLife), previously evaluated in clinical studies, consists of 16 active ingredients that are monographed in the Homeopathic Pharmacopeia of United States (HPUS) and recognized for their accelerated healing properties (reduction in post-op pain). The aim of this study was to compare VEGA to chlorhexidine (CHX) in vitro on gingival fibroblast viability, survival at various concentrations, migration assay, proliferation activity, expression of both regenerative growth factors as well as inflammatory markers, and collagen synthesis. A 10-fold dilution of standard CHX (0.02%) led to cell death, whereas cell viability was significantly better in the VEGA group for all tested parameters. Furthermore, VEGA also induced significantly greater fibroblast migration and proliferation. CHX negatively impacted the cellular inflammatory response of gingival fibroblasts, and also led to a reduction in collagen synthesis (50% decrease). Findings from the present study provide support from basic laboratory experiments that validate the previous clinical studies supporting the use of the VEGA oral rinse on its superior biocompatibility and wound healing properties when compared to CHX.

Enhancement of bone marrow aspirate concentrate with local self-healing corticotomies

Bone marrow aspirate concentrate (BMAC) is a potentially useful biological product for bone regeneration. This study investigated whether BMAC can be enriched by local minor corticotomies. Five 4-month-old domestic pigs were used with each pig undergoing two minor corticotomies at one randomly-selected tibia. Two weeks after the operation, bone marrow was aspirated from both tibiae and processed into BMAC samples. The amount of mesenchymal stem cells (MSCs) and the concentration of several regenerative growth factors contained in BMAC, as well as the proliferative and osteogenic differentiation capacity of MSCs, were compared between the corticotomy and the control sides. Another four weeks later, healing of the corticotomies was evaluated by radiographic and histological methods. The results demonstrated that BMAC from the corticotomy side contained significantly more MSCs than the control side. MSCs from the corticotomy side also proliferated significantly faster and tended to have stronger osteogenic differentiation than those from the control side. In contrast, the protein concentration of TGF-β, BMP-2 and PDGF contained in BMAC was only minimally changed by the corticotomies. The corticotomies in all pigs healed uneventfully, showing complete obliteration of the corticotomy gaps on CT images. Comparison between the two sides showed that the corticotomy side had thicker and denser cortical bone and more abundant osteogenic cell differentiation than the control side. These findings suggest that the quantity and proliferative/osteogenic differentiation capacity of MSCs contained in local BMAC can be enhanced by minor corticotomies, and spontaneous healing of the corticotomy can be completed within 6 weeks of the operation.

Endothelial Senescence in Regenerative Lung Growth after Pneumonectomy

Impairment of lung regeneration and repair in aging animals contributes to the pathogenesis of aging‐associated diseases, including chronic obstructive pulmonary disease (COPD). Angiogenesis – the formation of new blood capillaries ‐ plays a key role in organ regeneration. We have reported that inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and that post‐PNX regenerative lung growth is inhibited in aged mice. While accumulation of senescent cells in aged adults is linked to the age‐related decline in regenerative ability, senescent cells also play important roles in injury repair and tissue remodeling. Here we investigate the role of endothelial cell (EC) senescence in regenerative lung growth after PNX in young vs. aged mice. The levels of the senescence marker, p16INK4A and a major angiogenic factor, angiopoietin2 (Ang2) transiently increase in ECs isolated from 2‐months (2M) old post‐PNX mouse lungs compared to those from sham‐operated control mice and decrease at the later stage. The baseline levels of p16INK4A are high in 24M old mouse lung ECs but post‐PNX transient induction of p16INK4A and Ang2 is inhibited in 24M old mouse lung ECs. Depletion of senescent cells from 2M old p16‐3MR mice by treating with ganciclovir suppresses post‐PNX lung growth. When ECs isolated from 2M old post‐PNX mouse lungs are mixed in the gel and implanted on the mouse lungs, vascular network formation is stimulated in the gel, while these effects are inhibited in the gel mixed with 24M old post‐PNX mouse lung ECs. These results suggest that post‐PNX transient induction of EC senescence is required for vascular morphogenesis after PNX. Deregulation of this mechanism may contribute to age‐dependent inhibition of regenerative lung growth. Understanding this mechanism will lead to the development of efficient strategy for aging‐associated lung diseases.Support or Funding InformationNIH R21AG054830, NIH R21AG062893, NIH R01HL142578, AHA 18TPA34170129

Smart and Regenerative Urban Growth: A Literature Network Analysis.

“Smart city”, “sustainable city”, “ubiquitous city”, “smart sustainable city”, “eco-city”, “regenerative city” are fuzzy concepts; they are established to mitigate the negative impact on urban growth while achieving economic, social, and environmental sustainability. This study presents the result of the literature network analysis exploring the state of the art in the concepts of smart and regenerative urban growth under urban metabolism framework. Heat-maps of impact citations, cutting-edge research on the topic, tip-top ideas, concepts, and theories are highlighted and revealed through VOSviewer bibliometrics based on a selection of 1686 documents acquired from Web of Science, for a timespan between 2010 and 2019. This study discloses that urban growth is a complex phenomenon that covers social, economic, and environmental aspects, and the overlaps between them, leading to a diverse range of concepts on urban development. In regards to our concepts of interest, smart, and regenerative urban growth, we see that there is an absence of conceptual contiguity since both concepts have been approached on an individual basis. This fact unveils the need to adopt a more holistic and interdisciplinary approach to urban planning and design, integrating these concepts to improve the quality of life and public health in urban areas.

Obesity in Vascular and Alveolar Morphogenesis after Pneumonectomy

Obese population is rapidly growing worldwide. Obesity is associated with chronic lung diseases including chronic obstructive pulmonary disease (COPD). Impairment of lung regeneration and repair contributes to the pathogenesis of COPD. Angiogenesis – the formation of new blood capillaries ‐ plays a key role in organ regeneration. Inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and obesity is accompanied by endothelial cell (EC) dysfunction. However, the effects of obesity on lung vascular and alveolar regeneration remain unclear. Compensatory lung growth and vascular and alveolar morphogenesis after PNX are suppressed in leptin‐deficient ob/ob mice treated with high‐fat diet compared to that in control lean mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the brown adipose tissue collected from post‐PNX mice compared to those from sham‐operated control mice, while these effects are attenuated in post‐PNX leptin‐deficient ob/ob mice with high‐fat diet. The serum collected from post‐PNX control mice stimulates EC sprouting, while EC sprouting is inhibited when treated with serum collected from leptin‐deficient ob/ob mice after PNX. These results suggest that obesity may inhibit post‐PNX regenerative lung growth through VEGF signaling. Modulation of VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese population.Support or Funding InformationNIH R01HL142578

Mechanosensitive Mechanism of Lung Regeneration through Endothelial YAP1

Angiogenesis, the formation of new blood capillaries, plays a key role in organ development, homeostasis and regeneration. In addition to chemical factors, mechanical forces control angiogenesis and organ regeneration. We have reported that the mechanosensitive transcriptional co‐activator Yes‐associated protein (YAP1) in endothelial cells (ECs) is necessary for post‐pneumonectomy (PNX) lung growth. Right pulmonary artery (PA) pressure and shear stress increase after left unilateral PNX. However, it remains unknown whether changes in PA pressure and shear stress after PNX control post‐PNX regenerative lung growth through endothelial YAP1 signaling. Here we found that ligation of the right cardiac lobe PA after left PNX further increases right PA pressure and shear stress, upregulates expression of YAP1 and angiogenic factor Tie2, and stimulates post‐PNX lung growth in the remaining non‐occluded lobes. These effects are attenuated in VE‐cadherin‐specific Yap1 knockout mice. Hydrostatic pressure or shear stress applied to cultured human pulmonary arterial ECs also increases the expression and activity of YAP1 and induces EC proliferation and migration in vitro. These results suggest that increases in the right PA pressure together with elevation in shear stress after left PNX stimulate angiogenesis and post‐PNX lung growth through endothelial YAP1. Modulation of the mechanical environment or endothelial YAP1 expression/activity could be an efficient strategy for regenerative lung growth.Support or Funding InformationNIH RO1HL142578

It is not just a 'trade-off': indications for sink- and source-limitation to vegetative and regenerative growth in an old-growth beech forest.

Controls on tree growth are key issues in plant physiology. The hypothesis of our study was that the interannual variability of wood and fruit production are primarily controlled directly by weather conditions (sink limitation), while carbon assimilation (source limitation) plays a secondary role. We analyzed the interannual variability of weather conditions, gross primary productivity (GPP) and net primary productivity (NPP) of wood and fruits of an old-growth, unmanaged Fagus sylvatica forest over 14yr, including six mast years. In a multiple linear regression model, c. 71% of the annual variation in wood-NPP could be explained by mean air temperature in May, precipitation from April to May (positive influence) and fruit-NPP (negative influence). GPP of June to July solely explained c. 42% of the variation in wood-NPP. Fruit-NPP was positively related to summer precipitation 2yr before (R2 =0.85), and negatively to precipitation in May (R2 =0.83) in the fruit years. GPP had no influence on fruit-NPP. Our results suggest a complex system of sink and source limitations to tree growth driven by weather conditions and going beyond a simple carbon-mediated 'trade-off' between regenerative and vegetative growth.

Axon microdissection and transcriptome profiling reveals the in vivo RNA content of fully differentiated myelinated motor axons.

Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA).

A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration

Glial signals are known to inhibit axonal regeneration and functional recovery after mammalian central nervous system trauma, including spinal cord injury. Such signals include membrane-associated proteins of the oligodendrocyte plasma membrane and astrocyte-derived, matrix-associated proteins. Here, using cell lines and primary cortical neuron cultures, recombinant protein expression, immunoprecipitation and immunoblot assays, transmission EM of exosomes, and axon regeneration assays, we explored the secretion and activity of the myelin-associated neurite outgrowth inhibitor Nogo-A and observed exosomal release of a 24-kDa C-terminal Nogo-A fragment from cultured cells. We found that the cleavage site in this 1192-amino-acid-long fragment is located between amino acids 961-971. We also detected a Nogo-66 receptor (NgR1)-interacting Nogo-66 domain on the exosome surface. Enzyme inhibitor treatment and siRNA knockdown revealed that β-secretase 1 (BACE1) is the protease responsible for Nogo-A cleavage. Functionally, exosomes with the Nogo-66 domain on their surface potently inhibited axonal regeneration of mechanically injured cerebral cortex neurons from mice. Production of this fragment was observed in the exosomal fraction from neuronal tissue lysates after spinal cord crush injury of mice. We also noted that, relative to the exosomal marker Alix, a Nogo-immunoreactive, 24-kDa protein is enriched in exosomes 2-fold after injury. We conclude that membrane-associated Nogo-A produced in oligodendrocytes is processed proteolytically by BACE1, is released via exosomes, and is a potent diffusible inhibitor of regenerative growth in NgR1-expressing axons.

Macrophage Phenotype and Function in Liver Disorder.

Hepatic macrophages are a remarkably heterogeneous population consisting of self-renewing tissue-resident phagocytes, termed Kupffer cells (KCs), and recruited macrophages derived from peritoneal cavity as well as the bone marrow. KCs are located in the liver sinusoid where they scavenge the microbe from the portal vein to maintain liver homeostasis. Liver injury may trigger hepatic recruitment of peritoneal macrophages and monocyte-derived macrophages. Studies describing macrophage accumulation have shown that hepatic macrophages are involved in the initiation and progression of various liver diseases. They act as tolerogenic antigen-presenting cells to inhibit T-cell activation by producing distinct sets of cytokines, chemokines, and mediators to maintain or resolve inflammation. Furthermore, by releasing regenerative growth factors, matrix metalloproteinase arginase, they promote tissue repair. Recent experiments found that KCs and recruited macrophages may play different roles in the development of liver disease. Given that hepatic macrophages are considerably plastic populations, their phenotypes and functions are likely switching along disease progression. In this review, we summarize current knowledge about the role of tissue-resident macrophages and recruited macrophages in pathogenesis of alcoholic liver disease (ALD), non-alcoholic steatohepatitis (NASH), viral hepatitis, and hepatocellular carcinoma (HCC).

Nano-hydroxyapatite(n-HA) involved in the regeneration of rat nerve injury triggered by overloading stretch

Damage of axon and glial scars formation both inhibit nerve regenerative growth during nerve injury. In addition, mechanical stretch at high displacement rates of 10% tensile strain can cause marked nerve injury, it is important for finding a proper nano biomaterial to repair nerve injury. Nano-hydroxyapatite (n-HA) has excellent biocompatibility and high bioactivity, which is a good candidate for biomedical engineering applications. But the certain mechanism of n-HA on the injured nerve is seldom reported. In this study, we determined the role of n-HA on the mechanical stretch-induced nerve injury at adult rat spine. Mechanical stretch under strain 10% at displacement rates of 60 ​mm/min can cause marked broken vessels and edema in spinal cord and dorsal root ganglion tissue in haematoxylin-eosin (HE) staining. However, n-HA application can reverse hemorrhage and edema triggered by high rates of 60 ​mm/min stretch. Moreover, n-HA can promote positive staining of Netrin-1 increase significantly in spinal cord and dorsal root ganglion tested by immunohistochemistry (IHC) staining. In general, our study indicated that n-HA can repair mechanical stretch-induced nerve injury, it may provide a new approach to block injury and accelerate nerve regeneration in future.

Transcriptional analysis of scar-free wound healing during early stages of tail regeneration in the green anole lizard, Anolis carolinensis

While tail regeneration is observed in a number of vertebrate groups, including teleost fish such as the zebrafish, urodeles such as the axolotl, and anurans such as Xenopus tadpoles, mammalian and avian amniote vertebrates have lost this capacity. Among the amniotes, squamate reptiles such as lizards retain the ability to regrow their tails and also display the capacity to autotomize, or self-amputate, these structures as a predator evasion response. The regenerated tail is a biomechanically functional structure consisting of regrown and repatterned tissues including spinal cord, peripheral nerves, cartilage, skeletal muscle, vasculature, and skin. The green anole lizard, Anolis carolinensis, was the first reptile with a sequenced and annotated genome, thus allowing transcriptomic analyses. Furthermore, anoles exhibit a high degree of conservation of both innate and adaptive immune pathways with mammals. In histological analyses of tail regeneration in the green anole, we observed early cellular infiltration of the tail stump followed by a second phase of epithelial formation of the wound surface. These events preceded the period of rapid tail outgrowth, which typically starts at 10 days post autotomy. To identify genes activated during the initial phase of tail regeneration, we carried out whole transcriptome sequencing at 0.5, 1, 2, 3, 4, and 5 days post-autotomy. We identified that 5315 genes were differentially expressed between any of these time points and clustered into two major groups with elevated expression either in a first phase (0.5–1 DPA) or in a later second phase (3–5 DPA), with a marked shift in expression at 2 DPA. Genes with elevated expression in the first phase included those regulating the immune system, T cell receptor signaling, and the p38 MAPK signaling pathway. Genes upregulated in the second phase included those regulating cell proliferation, developmental growth, and Wnt/Hippo signaling pathways. Identifying the immunomodulatory events that set the stage for regenerative cell proliferation and outgrowth in an amniote model may help guide post-injury treatments as part of regenerative medical therapies.

Single-session cortical electrical stimulation enhances the efficacy of rehabilitative motor training after spinal cord injury in rats

Low neuronal cAMP levels in adults and a further decline following traumatic central nervous system (CNS) injury has been associated with the limited ability of neurons to regenerate. An approach to increase neuronal cAMP levels post injury is electrical stimulation. Stimulation as a tool to promote neuronal growth has largely been studied in the peripheral nervous system or in spared fibers of the CNS and this research suggests that a single session of electrical stimulation is sufficient to initiate a long-lasting axonal growth program. Here, we sought to promote plasticity and growth of the injured corticospinal tract with electrical cortical stimulation immediately after its spinal injury. Moreover, given the importance of rehabilitative motor training in the clinical setting and in translating plasticity into functional recovery, we applied training as a standard treatment to all rats (i.e., with or without electrical stimulation). Our findings show that electrical cortical stimulation did improve recovery in forelimb function compared to the recovery in unstimulated animals. This recovery is likely linked to increased corticospinal tract plasticity as evidenced by a significant increase in sprouting of collaterals above the lesion site, but not to increased regenerative growth through the lesion itself.

Cryopreserved human umbilical cord versus acellular dermal matrix patches for in utero fetal spina bifida repair in a pregnant rat model.

Despite significant improvement in spinal cord function after in utero spina bifida (SB) repair compared with traditional postnatal repair, over half of the children who undergo this procedure do not benefit completely. This lack of benefit has been attributed to closure methods of the defect, with subsequent spinal cord tethering at the repair site. Hence, a regenerative patch or material with antiinflammatory and anti-scarring properties may alleviate comorbidities with improved outcomes. The authors' primary objective was therefore to compare cryopreserved human umbilical cord (HUC) versus acellular dermal matrix (ADM) patches for regenerative repair of in utero SB lesions in an animal model. In vivo studies were conducted in retinoic acid-induced SB defects in fetuses of Sprague-Dawley rats. HUC or ADM patches were sutured over the SB defects at a gestational age of 20 days. Repaired SB defect tissues were harvested after 48-52 hours. Tissue sections were immunofluorescently stained for the presence of neutrophils, macrophages, keratinocytes, meningeal cells, and astrocytes and for any associated apoptosis. In vitro meningeal or keratinocyte cell coculture experiments with the ADM and HUC patches were performed. All experiments were scored quantitatively in a blinded manner. Neutrophil counts and apoptotic cells were lower in the HUC-based repair group (n = 8) than in the ADM patch repair group (n = 7). In the HUC patch repair group, keratinocytes were present on the outer surface of the patch, meningeal cells were present on the inner surface of the patch adjacent to the neural placode, and astrocytes were noted to be absent. In the ADM patch repair group, all 3 cell types were present on both surfaces of the patch. In vitro studies showed that human meningeal cells grew preferentially on the mesenchymal side of the HUC patch, whereas keratinocytes showed tropism for the epithelial side, suggesting an inherent HUC-based cell polarity. In contrast, the ADM patch studies showed no polarity and decreased cellular infiltration. The HUC patch demonstrated reduced acute inflammation and apoptosis together with superior organization in regenerative cellular growth when compared with the ADM patch, and is therefore likely the better patch material for in utero SB defect repair. These properties may make the HUC biomaterial useful as a "meningeal patch" during spinal cord surgeries, thereby potentially reducing tethering and improving on spinal cord function.

Recent advancements in understanding fin regeneration in zebrafish

Zebrafish have the remarkable ability to fully regenerate a lost appendage, faithfully restoring its size, shape and tissue patterning. Studies over the past decades have identified mechanisms underlying the formation, spatial organization, and regenerative growth of the blastema, a pool of proliferative progenitor cells. The patterning of newly forming tissue is tightly regulated to ensure proper rebuilding of anatomy. Precise niche regulation of retinoic acid and sonic hedgehog signaling ensures adherence to ray-interray boundaries. The molecular underpinnings of systems underlying re-establishment of pre-amputation size and shape (positional information) are also slowly starting to emerge. Osteoblasts play an important role as a cellular source of regenerating skeletal elements, and in zebrafish both osteoblast dedifferentiation as well as de novo osteoblast formation occurs. Both dedifferentiation and proliferation are tightly controlled, which makes it interesting to compare it to tumorigenesis, and to identify potential players involved in these processes. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

AAVshRNA-mediated PTEN knockdown in adult neurons attenuates activity-dependent immediate early gene induction

Genetic deletion or knockdown of PTEN enables regeneration of CNS axons, enhances sprouting of intact axons after injury, and induces de novo growth of uninjured adult neurons. It is unknown, however how PTEN deletion in mature neurons alters neuronal physiology. As a first step to address this question, we used immunocytochemistry for activity-dependent markers to assess consequences of PTEN knockdown in cortical neurons and granule cells of the dentate gyrus. In adult rats that received unilateral intra-cortical injections of AAV expressing shRNA against PTEN, immunostaining for c-fos under resting conditions (home cage, HC) and after 1 h of exploration of a novel enriched environment (EE) revealed no hot spots of c-fos expression that would suggest abnormal activity. Counts revealed similar numbers of c-fos positive neurons in the area of PTEN deletion vs. homologous areas in the contralateral cortex in the HC and similar induction of c-fos with EE. However, IEG induction in response to high frequency stimulation (HFS) of the cortex was attenuated in areas of PTEN deletion. In rats with AAVshRNA-mediated PTEN deletion in the dentate gyrus, induction of the IEGs c-fos and Arc with HFS of the perforant path was abrogated in areas of PTEN deletion. Immunostaining using phosphospecific antibodies for phospho-S6 (a downstream marker for mTOR activation) and phospho-ERK1/2 revealed abrogation of S6 phosphorylation in PTEN-deleted areas but preserved activation of phosphorylation of ERK1/2. Significance statementDeletion or knockdown of the tumor suppressor gene PTEN enables regenerative growth of adult CNS axons after injury, which is accompanied by enhanced recovery of function. Consequently, PTEN represents a potential target for therapeutic interventions to enhance recovery after CNS injury. Here we show that activity-dependent IEG induction is attenuated in PTEN-depleted neurons. These findings raise the intriguing possibility that functional recovery due to regenerative growth may be limited by the disruption of plasticity-related signaling pathways, and that recovery might be enhanced by restoring PTEN expression after regenerative growth has been achieved.

Cellular reprogramming for successful CNS axon regeneration is driven by a temporally changing cast of transcription factors

In contrast to mammals, adult fish display a remarkable ability to fully regenerate central nervous system (CNS) axons, enabling functional recovery from CNS injury. Both fish and mammals normally undergo a developmental downregulation of axon growth activity as neurons mature. Fish are able to undergo damage-induced “reprogramming” through re-expression of genes necessary for axon growth and guidance, however, the gene regulatory mechanisms remain unknown. Here we present the first comprehensive analysis of gene regulatory reprogramming in zebrafish retinal ganglion cells at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analyses reveal a regeneration program characterized by sequential activation of stage-specific pathways, regulated by a temporally changing cast of transcription factors that bind to stably accessible DNA regulatory regions. Strikingly, we also find a discrete set of regulatory regions that change in accessibility, consistent with higher-order changes in chromatin organization that mark (1) the beginning of regenerative axon growth in the optic nerve, and (2) the re-establishment of synaptic connections in the brain. Together, these data provide valuable insight into the regulatory logic driving successful vertebrate CNS axon regeneration, revealing key gene regulatory candidates for therapeutic development.

Inhibition of mmp13a during zebrafish fin regeneration disrupts fin growth, osteoblasts differentiation, and Laminin organization.

Matrix metalloproteinases 13 (MMP13) is a potent endopeptidase that regulate cell growth, migration, and extracellular matrix remodeling. However, its role in fin regeneration remains unclear. mmp13a expression is strongly upregulated during blastema formation and persists in the distal blastema. mmp13a knockdown via morpholino electroporation impairs regenerative outgrowth by decreasing cell proliferation, which correlates with a downregulation of fgf10a and sall4 expression in the blastema. Laminin distribution in the basement membrane is also affected in mmp13a MO-injected rays. Another impact of mmp13a knockdown is observed in the skeletal elements of the fin rays. Expression of two main components of actinotrichia, Collagen II and Actinodin 1 is highly reduced in mmp13a MO-injected rays leading to highly disorganized actinotrichia pattern. Inhibition of mmp13a strongly affects bone formation as shown by a reduction of Zns5 and sp7 expression and of bone matrix mineralization in rays. These defects are accompanied by a significant increase in apoptosis in mmp13a MO-injected fin regenerates. Defects of expression of this multifunctional proteinase drastically affects osteoblast differentiation, bone and actinotrichia formation as well as Laminin distribution in the basement membrane of the fin regenerate, suggesting the important role of Mmp13 during the regenerative process.

Anisotropy Properties of Tissues: A Basis for Fabrication of Biomimetic Anisotropic Scaffolds for Tissue Engineering

Tissue engineering has been a subject of extensive scientific exploration in the last two decades making gradual inroads into clinical studies as well. Along with regenerative cells and growth factors, biomaterial scaffolds are integral to the development of a tissue engineered construct. It is now appreciated that scaffolds should mimic the target tissue properties intimately in order to provide a micro-environment milieu that allows the seeded cells to differentiate into the desired tissue. Even from a structural viewpoint, mismatch between scaffold and native matrix properties can cause cell necrosis through mechanisms such as stress shielding. One of the key properties of most body tissues is that they exhibit anisotropy. However, most fabrication methods generate isotropic scaffolds and require specific modifications to produce anisotropic scaffolds. In the last decade, the advent of additive manufacturing and bioprinting has provided facile tools to fabricate scaffolds with desired anisotropy. On the other hand, a biomimetic scaffold can be designed only when target tissue anisotropy is well known to the tissue engineer. This review presents an overview of the anisotropic properties of different tissues, which will be critical for developing biomimetic engineered constructs. The traditional anatomical records do not adequately present these properties from the perspective of designing tissue engineering scaffolds. Subsequently, present state-of-the art in development of anisotropic scaffolds as well as tissue constructs using different conventional and emerging fabrication techniques is discussed. It is expected that the readers will obtain a comprehensive reference on the research area by examining these two aspects juxtaposed to each other and gain key trends for fabrication of anisotropic scaffolds, plausibly with improved regenerative outcomes.

PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.

The molecular mechanisms discriminating between regenerative failure and success remain elusive. While a regeneration-competent peripheral nerve injury mounts a regenerative gene expression response in bipolar dorsal root ganglia (DRG) sensory neurons, a regeneration-incompetent central spinal cord injury does not. This dichotomic response offers a unique opportunity to investigate the fundamental biological mechanisms underpinning regenerative ability. Following a pharmacological screen with small-molecule inhibitors targeting key epigenetic enzymes in DRG neurons, we identified HDAC3 signalling as a novel candidate brake to axonal regenerative growth. In vivo, we determined that only a regenerative peripheral but not a central spinal injury induces an increase in calcium, which activates protein phosphatase 4 that in turn dephosphorylates HDAC3, thus impairing its activity and enhancing histone acetylation. Bioinformatics analysis of exvivo H3K9ac ChIPseq and RNAseq from DRG followed by promoter acetylation and protein expression studies implicated HDAC3 in the regulation of multiple regenerative pathways. Finally, genetic or pharmacological HDAC3 inhibition overcame regenerative failure of sensory axons following spinal cord injury. Together, these data indicate that PP4-dependent HDAC3 dephosphorylation discriminates between axonal regeneration and regenerative failure.