Physiological Wound Healing Research Articles

Potato Periderm is the First Layer of Defence against Biotic and Abiotic Stresses: a Review

The frequently changing climate has resulted in plants facing more biotic and abiotic stresses than ever before, leading to yield and quality losses. The global population is increasing at an alarming rate and feeding the world has become a huge challenge. Being a staple crop of many countries, potato can help to overcome this challenge. A comprehensive knowledge of physiology, biochemistry and molecular genetics of development and wound healing of potato tuber periderm (skin) is very important as the periderm protects the tubers from skinning injuries, dehydration, bruising and biotic and abiotic stresses. Improvements in tuber periderm are essential to enhance tuber quality and shelf life and reduce financial losses. In this paper, we have reviewed the protective roles of tuber periderm layer in response to stress and injuries. Moreover, molecular regulation of native and wound-induced periderm formation has been discussed. Since suberization is an important aspect of tuber periderm formation and wound healing, we have also reviewed the key genes involved in this process. This review will pave the way for obtaining potato tubers resistant to various abiotic and pathogenic stresses.

Skin Wound Healing Process and New Emerging Technologies for Skin Wound Care and Regeneration.

Skin wound healing shows an extraordinary cellular function mechanism, unique in nature and involving the interaction of several cells, growth factors and cytokines. Physiological wound healing restores tissue integrity, but in many cases the process is limited to wound repair. Ongoing studies aim to obtain more effective wound therapies with the intention of reducing inpatient costs, providing long-term relief and effective scar healing. The main goal of this comprehensive review is to focus on the progress in wound medication and how it has evolved over the years. The main complications related to the healing process and the clinical management of chronic wounds are described in the review. Moreover, advanced treatment strategies for skin regeneration and experimental techniques for cellular engineering and skin tissue engineering are addressed. Emerging skin regeneration techniques involving scaffolds activated with growth factors, bioactive molecules and genetically modified cells are exploited to overcome wound healing technology limitations and to implement personalized therapy design.

Artificial dermal substitutes for tissue regeneration: comparison of the clinical outcomes and histological findings of two templates.

ObjectiveArtificial dermal substitutes (DSs) are fundamental in physiological wound healing to ensure consistent and enduring wound closure and provide a suitable scaffold to repair tissue. We compared the clinical and histological features of two DSs, Pelnac and Integra, in the treatment of traumatic and iatrogenic skin defects.MethodsThis prospective observational study involved 71 randomly selected patients from our hospital. Wound healing was analyzed using the Wound Surface Area Assessment, the Vancouver Scar Scale, and a visual analog scale. Histological and immunohistochemical evaluations were also performed.ResultsAt 2 weeks, greater regeneration with respect to proliferation of the epidermis and renewal of the dermis was observed with Pelnac than with Integra. At 4 weeks, the dermis had regenerated with both DSs. Both templates induced renewed collagen and revascularization. Differences in the Vancouver Scar Scale score were statistically significant at 4 weeks and 1 year. Pelnac produced a significant increase in contraction at 2 weeks with increasing effectiveness at 4 weeks. Integra produced a higher percentage reduction in the wound surface area and a shorter healing time than Pelnac for wounds >1.5 cm deep.ConclusionOur observational data indicate that both DSs are effective and applicable in different clinical contexts.

WOUND HEALING CONCEPTS: CONTEMPORARY PRACTICES AND FUTURE PERSPECTIVES

The advancements in the development of wound dressings have seen tremendous growth in the past few decades. Wound healing approach has majorly shifted from dry healing to moist healing. There has been a significant advancement in our understanding of the underlying physiology involved in wound healing and the associated systemic factors having a direct or indirect influence on the healing. This has resulted in the development of wound dressings designed to treat specific types of wounds. The present review discusses the physiology of wound healing, followed by different factors that contribute to healing. The advancements in wound dressings with their merits and limitations, newer approaches in wound care i.e., hyperbaric oxygen, negative pressure therapy, skin substitutes and role of growth factors in wound healing, have been highlighted. In addition, more recent approaches for effective wound care like smart devices with sensing, reporting and responding functions are discussed.

Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces.

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet's periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.

Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.

Multinucleated giant cells within the in vivo implantation bed of a collagen-based biomaterial determine its degradation pattern

ObjectivesThe aim of the present study was to characterize the cellular reaction to a xenogeneic resorbable collagen membrane of porcine origin using a subcutaneous implantation model in Wistar rats over 30 days.Materials and methodsEx vivo, liquid platelet-rich fibrin (PRF), a leukocyte and platelet-rich cell suspension, was used to evaluate the blood cell membrane interaction. The material was implanted subcutaneously in rats. Sham-operated rats without biomaterial displayed physiological wound healing (control group). Histological, immunohistological, and histomorphometric analyses were focused on the inflammatory pattern, vascularization rate, and degradation pattern.ResultsThe membrane induced a large number of mononuclear cells over the observation period, including lymphocytes, macrophages, and fibroblasts. After 15 days, multinucleated giant cells (MNGCs) were observed on the biomaterial surface. Their number increased significantly, and they proceeded to the center of the biomaterial on day 30. These cells highly expressed CD-68, calcitonin receptor, and MMP-9, but not TRAP or integrin-ß3. Thus, the membrane lost its integrity and underwent disintegration as a consequence of the induction of MNGCs. The significant increase in MNGC number correlated with a high rate of vascularization, which was significantly higher than the control group. Physiological wound healing in the control group did not induce any MNGCs at any time point. Ex vivo blood cells from liquid-PRF did not penetrate the membrane.ConclusionThe present study suggests a potential role for MNGCs in biomaterial degradation and questions whether it is beneficial to accept them in clinically approved biomaterials or focus on biomaterials that induce only mononuclear cells. Thus, further studies are necessary to identify the function of biomaterial-induced MNGCs.Clinical relevanceUnderstanding the cellular reaction to biomaterials is essential to assess their suitability for specific clinical indications and outline the potential benefit of specific group of biomaterials in the respective clinical indications.

Call for Special Issue Papers: Methodology of Research and Applications of Electric Fields

BioelectricityVol. 2, No. 2 Calls for PapersFree AccessCall for Special Issue Papers: Methodology of Research and Applications of Electric FieldsDeadline for Manuscript Submission: July 15, 2020Guest Editors: Min Zhao, Ann Rajnicek, and Francis LinGuest Editors: Min ZhaoUniversity of California, Davis.Search for more papers by this author, Ann RajnicekUniversity of Aberdeen.Search for more papers by this author, and Francis LinUniversity of Manitoba.Search for more papers by this authorPublished Online:17 Jun 2020https://doi.org/10.1089/bioe.2020.29013.cfp2AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Bioelectricity is central to the physiology and pathology of wound healing, nervous system function, immune responses, cardiac and muscle functions, and cancers. Recent technological advances in engineering, physics, chemistry and computer science have spawned wide-ranging electric field-based methodologies with biological and biomedical applications. These continue to emerge, so this timely special issue aims to be a focal point for innovation. We now solicit high quality original research papers on novel research methodologies and applications of electric fields. Other types of manuscripts will also be considered.Topics can include (but are not restricted to) microfluidic devices for research applications of biomolecules, cells and microorganisms mediated by electric fields, modeling and computational methods for studying bioelectricity in physiological and pathological systems or in in-vitro models, electrode fabrication and electrode array designs to enable new bio-applications, development of novel devices utilizing electrical phenomena to diagnose or treat disease. Scientists, engineers, clinicians and other professionals who are actively pursuing research relevant to this special issue are strongly encouraged to submit their work.Bioelectricity will consider articles from the full breadth of evidence: from original research papers and reviews, to perspectives, profiles, commentaries, and exemplary front matter.Areas of interest include, but are not limited to: Topic A: Microfluidic devices for research applications of biomolecules, cells and other microorganisms mediated by electric fieldsTopic B: Modeling and computational methods for studying bioelectricity in physiological and pathological systems or in in-vitro modelsTopic C: Electrode fabrication and electrode array designs to enable novel bio-applicationsTopic D: Development of novel diagnostic or therapeutic devices utilizing electrical phenomena to diagnose or treat disease.Contributions will receive prompt and thorough peer review. Please refer to our Instructions for Authors at www.liebertpub.com/bioe before submitting your manuscript for consideration. Authors are encouraged to vet ideas directly with Dr. Francis Lin via e-mail before submitting (francis.lin@umanitoba.ca).Bioelectricity is the only journal devoted explicitly to bioelectricity research. The field of bioelectricity, much like the field of biochemistry, is becoming a foundational component of the biological sciences. The scientists who pioneered modern studies of bioelectricity have created a solid footing for the imminent upsurge in exploration: voltage, current, resistance, pH, and other familiar ion-dependent phenomena, have all been found to be generated and maintained by cells in highly conserved, energy-dependent processes. Spearheaded by pioneering co-Editors-in-Chief, Mustafa Djamgoz, PhD, and Michael Levin, PhD, Bioelectricity is published online and in print with open access options and rapid turnaround of submissions. The Journal provides a high-profile, international forum for groundbreaking original research papers, review articles, captivating front matter, debate, and commentary. Bioelectricity is an exciting and dynamic focal point for the growing bioelectricity community and is the go-to resource that will drive the field forward.Editorial questions? Please contact Dr. Francis Lin (francis.lin@umanitoba.ca).Visit the Instructions for Authors:www.liebertpub.com/bioeSubmit your paper for peer review online:https://mc.manuscriptcentral.com/bioeFiguresReferencesRelatedDetails Volume 2Issue 2Jun 2020 InformationCopyright 2020, Mary Ann Liebert, Inc., publishersTo cite this article:Guest Editors: Min Zhao, Ann Rajnicek, and Francis Lin.Bioelectricity.Jun 2020.65-65.http://doi.org/10.1089/bioe.2020.29013.cfp2Published in Volume: 2 Issue 2: June 17, 2020PDF download

Mutual inter-regulation between iNOS and TGF-β1: Possible molecular and cellular mechanisms of iNOS in wound healing

Abnormal wound healing with excessive scarring is a major health problem with socioeconomic and psychological impacts. In human, chronic wounds and scarring are associated with upregulation of the inducible nitric oxide synthase (iNOS). Recently, we have shown physiological regulation of iNOS in wound healing. Here, we sought to investigate the possible mechanistic role of iNOS in wound healing using biochemical and immunohistochemical assays. We found: (a) iNOS is the main source of wound nitric oxide (NO), (b) NOS inhibition in the wound, downregulated iNOS protein, mRNA and enzymatic activity, and reduced wound NO, and (c) iNOS inhibition resulted in delayed healing at early time points, and excessive scarring at late time points. Furthermore, molecular and cellular analysis of the wound showed that iNOS inhibition significantly (P < 0.05) increased TGF-β1 mRNA and protein levels, fibroblasts and collagen deposition. These latter findings suggest that iNOS might be exerting its action in the wound by signaling through TGF-β1 that activates wound fibroblasts to produce excessive collagen. Our current findings provide further support that iNOS is crucial for physiological wound healing, and suggest that dysregulation of iNOS during the inflammatory phase impairs healing, and results in disfiguring post-healing scarring. Thus, the mutual feedback regulation between iNOS and TGF-β1 at the gene, protein and functional levels might be the mechanism through which iNOS regulates the healing. Monitoring and maintenance of wound NO levels might be important for healing and avoiding long-term complications in susceptible people including patients with diabetic wounds, venous ulcers or keloid prone.

589-P: Impaired Endothelial Nrf2 Expression Reduces Wound Neovascularization in Diabetic Mice

The global cost of diabetes in the U.S. is 1.3 trillion dollars, with chronic diabetic foot ulcers accounting for up to 13 billion dollars annually. An underlying factor that contributes to the chronicity of these wounds is poor vascular flow, which impedes physiologic wound healing. Here, using an in vivo murine model of wound healing, we demonstrate that Nrf2 expression in endothelial cells is necessary for physiologic neovascularization. Full-thickness 10 mm stented excisional wounds were created on the dorsa of double transgenic mouse with inducible conditional deletion of Nrf2 in cadherin 5 expressing endothelial cells (KO), wildtype (WT), and heterozygous mice. Time to wound closure was delayed in the KO compared to WT mice (KO 33.0±1.73 days vs. WT 14.0±0 days) and Nrf2 KO healed similarly to diabetic mice (KO 33.0±1.73 days vs. Diabetic 31.0±1.41 days). Endothelial cells isolated from the wounds of KO mice via flow cytometry demonstrated significantly decreased angiogenic functionality when compared to heterozygous controls. KO endothelial cells formed significantly fewer branches (KO 64.75±14.81 branches/cm2 vs. control 282.8±46.7 branches/cm2, p=0.014) and fewer branching points (KO 39.0±5.18 branching points/cm2 vs. Control 150.8±15.34 branching points/cm2, p=0.0031), indicating impaired angiogenic activity. Immunofluorescent staining of endothelial cells within the wound beds of KO also demonstrate decreased vascularity compared to WT controls. We looked at regulation of the CXCR4/SDF-1 axis, key to angiogenesis, using C166 mouse endothelial cells. Nrf2 knockdown cells in hypoxia, results in reduced Nrf2 levels by 76.5% (p=0.001) and that of downstream marker NQO1 by 66.4% (p=0.02). Expression of CXCR4 and SDF-1 decreased 61.7% (p=0.001) and 47.6% (p=0.02) respectively. These findings suggest that the Nrf2 dysregulation associated with diabetes may contribute to the impaired angiogenesis and decreased revascularization present in chronic diabetic wounds. Disclosure J. Lee: None. A.P. Villarreal Ponce: None. J.A. David: None. J. Kuhn: None. J. Kwong: None. D. Ceradini: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-ACE-08 to D.C.)

Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces.

Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet's periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.

HUMAN ALLOGRAFTS IMPROVE HEALING OUTCOMES IN CHRONIC WOUNDS

BackgroundChronic wounds affect an estimated 8 million people worldwide and are often a result of underlying health conditions, such as obesity, diabetes, malnutrition, etc. The related morbidity and mortality places an increased burden on the healthcare system. Regenerative approaches, which engages and stimulates the body’s innate healing capabilities is addressing the limited successes in current standard of care treatments. Axolotl Biologix is a regenerative medicine company that produces allograft products from the human amnion. Axolotl Ambient™ and Shot™, conditioned media rich in cytokines and growth factors, are derived from human amniotic mesenchymal stromal cells. Axolotl DualGraft™ is a dehydrated, acellular, double layer, human amniotic membrane that encourages soft tissue repair by serving as a barrier, extracellular matrix scaffold, and provides a source of matrix‐bound growth factors.MethodsBoth allografts were used to facilitate repair and regeneration in a patient with difficult to close or intractable full‐thickness wounds. Axolotl Ambient™ was injected into the wound margin and Axolotl DualGraft™ was placed within the exposed wound bed. Wounds were bandaged per standard of care and imaged regularly to monitor wound healing progression. A range of bioassays were performed to ascertain the biological activity of the allografts.ResultsMultiple applications and injections of Ambient and DualGraft resulted in the formation of granulation tissue, restoration of blood flow, pain sensation, epithelialization, and wound closure. Bioassays confirmed the allografts contain measurable amounts of growth factors that promote angiogenesis, proliferation, tissue remodeling and reduction in inflammation.ConclusionsClinical application of the allografts in intractable wounds yielded dramatic improvement in wound repair by stimulating the normal physiological wound healing processes by the addition of exogenous growth factors, providing a scaffold in the wound bed, promoting normal blood flow, re‐epithelialization, and tissue remodeling. Longitudinal case studies are ongoing, concurrent with benchtop research to further elucidate the mechanisms of action responsible for reported clinical benefits in difficult to heal wounds.Support or Funding InformationN/A

Multifunctional Copper-Containing Mesoporous Glass Nanoparticles as Antibacterial and Proangiogenic Agents for Chronic Wounds.

The physiological wound healing process involves a cascade of events which could be affected by several factors resulting in chronic, non-healing wounds. The latter represent a great burden especially when bacterial biofilms are formed. The rise in antibiotic resistance amongst infectious microorganisms leads to the need of novel approaches to treat this clinical issue. In this context, the use of advanced biomaterials, which can enhance the physiological expression and secretion of the growth factors involved in the wound healing process, is gaining increasing attention as a robust and appealing alternative approach. Among them, mesoporous glasses are of particular interest due to their excellent textural properties and to the possibility of incorporating and releasing specific therapeutic species, such as metallic ions. One of the most attractive therapeutic ions is copper thanks to its proangiogenic and antibacterial effects. In this contribution, copper containing mesoporous glass nanoparticles were proposed as a multifunctional device to treat chronic wounds. The developed nanoparticles evidenced a very high specific surface area (740 m2/g), uniform pores of 4 nm and an almost total release of the therapeutic ion within 72 h of soaking. The produced nanoparticles were biocompatible and, when tested against Gram positive and Gram negative bacterial species, demonstrated antibacterial activity against both planktonic and biofilm bacteria in 2D cell monolayers, and in a 3D human model of infected skin. Their proangiogenic effect was tested with both the aortic ring and the chick chorioallantoic membrane assays and an increase in endothelial cell outgrowth at a concentration range between 30 and 300 ng/mL was shown. Overall, in this study biocompatible, multifunctional Cu-containing mesoporous glass nanoparticles were successfully produced and demonstrated to exert both antibacterial and proangiogenic effects.

646 Impact of Inflammation on Wound Healing

Abstract Introduction Inflammation is part of the physiological phases of wound healing. It serves the purpose to attract different immune cells in order to clean the wound from pathogens and debris and create an ideal environment for fibroblasts and keratinocytes to differentiate, migrate and divide in order to close the wound. Pathological inflammation caused by many different factors is often the underlying cause of prolonged wound healing and unfavourable outcome in burn patients. We wanted to investigate what influence an induced hyper-inflammation on wound healing has in a controlled environment. Methods In a porcine wound model, pigs received full-thickness wounds on their backs where hyper-inflammation was induced by application of a chemotherapeutic for 6 days. Control wounds were left without induction. Wound were scored on a daily basis for 16 days in total and biopsies were taken for RNA analysis as well as histological assessment. Results The results showed a rise in inflammation of the wounds and the surrounding tissue due to the induction. The wounds healed slower, needing up to seven more days to completely heal. The results of the RNA analysis showed high values of pro-inflammatory cytokines after the induction in comparison to the baseline and control wounds. Histological assessment showed thick layers of necrosis, adipocyte as well as leucocyte infiltration in the hyper-inflamed wounds compared to completely healed control wounds with all layers of epidermis visible. Conclusions Hyper-inflammation, even just for a short period of time, can significantly prolong the time of wound healing. These findings underline the importance of preventing and treating wound infections of burn patients in order to support physiological wound healing as well as to avert complications such as infections with multi-resistant microbes that are associated with prolonged hospital stays. Applicability of Research to Practice The findings of these experiments cannot be directly applied to practice however, the influence of inflammation should kept in mind when treating burn patients.

Lesional skin in vitiligo exhibits delayed in vivo reepithelialization compared to the nonlesional skin.

Vitiligo, a common skin disorder, is characterized by the loss of functional melanocytes resulting in the depigmentation of skin. Previous studies have demonstrated molecular and architectural alterations in the epidermal keratinocytes upon loss of melanocytes. The physiological implications of these "altered" keratinocytes are yet not known. We investigated the wound healing efficiency of lesional vs nonlesional skin in 12 subjects with stable nonsegmental vitiligo using histological and ultrastructural evaluation of partial-thickness wounds. The wounds were examined 12 days postinjury, coinciding with the reepithelialization phase of healing marked primarily by keratinocyte migration and proliferation. This study demonstrated a significant difference in the reepithelialization potential between the lesional and nonlesional skin. While all 12 nonlesional wounds demonstrated considerable neoepidermis formation on the 12th day post wound, only four of the corresponding lesional samples showed comparable reepithelialization; the rest remaining in the inflammatory phase. Ultrastructural studies using transmission electron microscopy as well as immunohistochemical staining revealed a reduced number of desmosomes, shorter keratin tonofilaments and an increase in myofibroblast population in the dermis of lesional reepithelialized tissue compared to the nonlesional reepithelialized samples. This study implicates gross functional perturbations in the lesional skin during physiological wound healing in vitiligo, suggesting that the breakdown of keratinocyte-melanocyte network results in delayed wound repair kinetics in the lesional skin when compared to patient-matched nonlesional skin.

Modification of collagen-based sponges can induce an upshift of the early inflammatory response and a chronic inflammatory reaction led by M1 macrophages: an in vivo study

BackgroundThe present study evaluated the cellular tissue reaction of two equine-derived collagen hemostatic sponges (E-CHS), which differed in thickness after pressing, over 30 days in vivo. The inflammatory response during physiological wound healing in sham-operated animals was used as control group.Material and methodsFirst, the E-CHS was pressed by applying constant pressure (6.47 ± 0.85 N) for 2 min using a sterile stainless-steel cylinder until the material was uniformly flattened. Consequently, the original (E-CHS), the pressed (P-E-CHS), as well as the control group (CG; sham operation) were studied independently. The 3 groups were evaluated in vivo after subcutaneous implantation in Wistar rats during 3, 15, and 30 days. Histochemical and immunohistochemical methods provided observations of biomaterial degradation rate, cellular inflammatory response, and vascularization pattern. A derivative of human blood known as platelet-rich fibrin (PRF) was used as an ex vivo model to simulate the initial biomaterial-cell interaction. Segments of E-CHS and P-E-CHS were cultivated for 3 and 6 days with PRF, and the release of pro-inflammatory proteins was measured using ELISA. PRF cultivated alone was used as a control group.ResultsAt day 3, the CG induced a statistically significant higher presence of monocytes/macrophages (CD68+), pro-inflammatory macrophages (M1; CCR7+), and pro-wound healing macrophages (M2; CD206+) compared to E-CHS and P-E-CHS. At the same time point, P-E-CHS induced a statistically significant higher presence of CD68+ cells compared to E-CHS. After 15 days, E-CHS was invaded by cells and vessels and showed a faster disintegration rate compared to P-E-CHS. On the contrary, cells and vessels were located only in the outer region of P-E-CHS and the biomaterial did not lose its structure and accordingly did not undergo disintegration. The experimental groups induced similar inflammatory reaction primarily with positive pro-inflammatory CD68+/CCR7+ macrophages and a low presence of multinucleated giant cells (MNGCs). At this time point, significantly lower CD68+/CCR7+ macrophages and no MNGCs were detected within the CG when compared to the experimental groups (P < 0.05). After 30 days, E-CHS and P-E-CHS were fully degraded. All groups showed similar inflammatory reaction shifted to a higher presence CD206+ macrophages. A low number of CCR7+ MNGCs were still observable in the implantation bed of both experimental groups. In the ex vivo model, the cells and fibrin from PRF penetrated E-CHS. However, in the case of P-E-CHS, the cells and fibrin stayed on the surface and did not penetrate towards materials central regions. The cultivation of P-E-CHS with PRF induced a statically significant higher release of pro-inflammatory proteins compared to the CG and E-CHS after 3 days.ConclusionAltering the original presentation of a hemostatic sponge biomaterial by pressing modified the initial biomaterial-cell interaction, delayed the early biomaterial’s degradation rate, and altered the vascularization pattern. A pressed biomaterial seems to induce a higher inflammatory reaction at early time points. However, altering the biomaterial did not modify the polarization pattern of macrophages compared to physiologic wound healing. The ex vivo model using PRF was shown to be an effective model to simulate the initial biomaterial-cell interaction in vivo.Clinical relevanceA pressed hemostatic sponge could be applied for guided tissue regeneration and guided bone regeneration. In that sense, within the limitations of this study, the results show that the same biomaterial may have two specific clinical indications.

Integrity of dural closure after autologous platelet rich fibrin augmentation: an in vitro study

BackgroundWatertight closure of the dura mater is fundamental in neurosurgery. Besides the classical suturing techniques, a variety of biomaterials have been proposed as sealants. Platelet rich fibrin (PRF) is an autologous biomaterial which can readily be obtained through low-speed centrifugation of patient’s own blood. It is rich in fibrin, growth factors, leucocytes and cytokines and has shown adhesive properties while promoting the physiological wound healing process. In this study, we investigated the effect of applying PRF in reinforcing the watertight dura mater closure.MethodsWe created an in vitro testing device, where the watertight dura mater closure could be hydrostatically assessed. On 26 fresh harvested bovine dura maters, a standardised 20-mm incision was closed with a running suture, and the leak pressure was measured first without (primary leak pressure) and then with PRF augmentation (secondary leak pressure). The two groups of measurements have been statistically analysed with the Student’s paired t test.ResultsThe “running suture only group” had a leak pressure of 10.5 ± 1.2 cmH2O (mean ± SD) while the “PRF-augmented group” had a leak pressure of 47.2 ± 2.6 cm H2O. This difference was statistically significant (p < 0.001; paired t test).ConclusionsAutologous platelet rich fibrin augmentation reliably reinforced watertight closure of the dura mater to a > 4-fold increased leak pressure after failure of the initial standard running suture technique.

Analysis of vascular smooth muscle cell and HeLa cell migration on the microgrooved substrate (Cell type differences of the mechanical sensing for microgrooved surfaces)

Cell migration is critical in many physiological processes, including morphogenesis, wound healing, and metastasis of cancer cells, and thus elucidation of the mechanism of cell migration is quite important. It is well known that these processes of cell migration can be guided by microenvironment of the substrate surfaces, such as microgrooves. However, detailed mechanism of cellular mechanical sensing for microgrooved surfaces has not been clarified yet. Here we investigated the migration responses of cells on the microgrooved substrate in consideration of the effects of cell type differences. We cultured vascular smooth muscle cells (VSMCs) obtained from different animal species (porcine aortic smooth muscle (PASM) cells and rat aortic smooth muscle (RASM) cells) and cervical cancer cells (HeLa) on the microgrooved substrate with 2-μm groove width and approximately 200-nm groove depth, and assessed the shape changes and migration behavior of the cells. Both types of VSMCs migrated directionally on the microgrooved substrate, but their detailed responses were quite different: PASM cells became more elongated shape on the microgrooves while maintaining their adhesion area and exhibited marked directional migration on the grooves. In contrast, RASM cells showed a significant reduction of adhesion area on the microgrooved substrate and they occasionally locomoted obliquely on the grooves. Interestingly, HeLa cells did not elongate in their shape on the microgrooved substrate with random migration direction, indicating that HeLa cells could not sense the microgrooved surfaces. These results indicate that the changes in cell shape and migration behavior might be dependent on the difference in intracellular structure due to the cell type differences, and these differences can be emphasized by the culture on the microgrooved surfaces.

Silver-impregnated platelet-rich fibrin as a barrier membrane

Introduction: Platelet-rich fibrin (PRF) was developed to eliminate xenofactors form of platelet-rich plasma to be used as a source of growth factor for tissue regeneration. PRF has been used as an autologous grafting material because of its ability to accelerate physiologic wound healing and new bone formation. Aim: To compare mechanical and histologic characteristics of the PRF membrane and silver-impregnated PRF. Materials and Methods: Venous blood was taken from the subjects for PRF preparation. Then, 2 normal PRFs were prepared, and for silver-impregnated PRF, 9 ml of blood in addition to 1 ml of silver nanoparticle suspension was poured into another tube and gently shook with hand to achieve a uniform 1% concentration. The tubes were centrifuged at 3000 rpm for 10 min. The tensile test was done by the universal testing machine: The remaining pieces of the membranes were fixed in 10% formalin for 24 h to be subjected to hematoxylin and eosin staining and evaluated with the light microscope Degradation time. Results: Silver-impregnated showed improved mechanical properties and dense fibrin network than PRF. Conclusion: Silver-impregnated PRF membrane demonstrated properties to be used as a barrier membrane for periodontal reconstruction.

Femtosecond Laser-Assisted Surgery for Implantation of Bioengineered Corneal Stroma to Promote Corneal Regeneration.

The femtosecond laser has achieved widespread use in ophthalmology owing to its ability to deliver focused high energy that is rapidly dissipated and thereby does not damage surrounding tissue outside the precise focal region. Extremely accurate and smooth cuts can be made by the laser, enabling a range of applications in anterior segment surgery. Minimally invasive corneal surgical procedures can be performed using the femtosecond laser, and here we describe the application of such procedures to improve implantation of bioengineered materials into the cornea. Bioengineered corneal tissue, including the collagenous corneal stroma, promises to provide a virtually unlimited supply of biocompatible tissue for treating multiple causes of corneal blindness globally, thereby circumventing problems of donor tissue shortages and access to tissue banking infrastructure. Optimal implantation of bioengineered materials, however, is required, in order to facilitate postoperative wound healing for the maintenance of corneal transparency and avoidance of postoperative complications such as scarring, inflammation, and neovascularization. Moreover, the avoidance of a detrimental physiological physiological wound healing response is critical for facilitating the corneal stromal regeneration enabled by the bioengineered stroma. Without proper implantation, the tissue response will favor inflammation and pathologic processes instead of quiescent keratocyte migration and new collagen production. Here we describe several procedures for optimized biomaterial implantation into the corneal stroma, that facilitate rapid wound healing and regenerative restoration of corneal transparency without the use of human donor tissue. A step-by-step methodology is provided for the use of the femtosecond laser and associated techniques, to enable seamless integration of bioengineered materials into the corneal stroma.