Use Of Fibroblasts Research Articles

Prospects for the application of cell therapy in combustiology

Currently, one of the urgent problems in the field of surgery and traumatology is burn injury. A significant part of the victims has deep and extensive burns, the treatment of which is the most difficult problem of modern combustiology. In this aspect, one of the main problems faced by surgeons in combustiology is the lack of donor material and an increased risk of complications of burn wounds. In this regard, the most promising direction in kombustiology is the application of cell engineering methods, including the use of fibroblasts and stem cells. Fibroblasts have a number of advantages, such as efficiency, safety and accessibility. Stem cells are also a common method of cell therapy, they have great potential in regenerative processes. Mesenchymal stem cells (MSCs), which have a pronounced regenerative and immunosuppressive effect, are also used in kombustiology. In the treatment of infected burn wounds, phage therapy, which provides lysis of infectious pathogens, showed a positive result. In addition to fighting pathogens, bacteriophages, like MSCs, have an immunosuppressive effect, thereby reducing the risk of transplant rejection.

The Role of Fibroblasts in Skin Homeostasis and Repair.

Fibroblasts are typical mesenchymal cells widely distributed throughout the human body where they (1) synthesise and maintain the extracellular matrix, ensuring the structural role of soft connective tissues; (2) secrete cytokines and growth factors; (3) communicate with each other and with other cell types, acting as signalling source for stem cell niches; and (4) are involved in tissue remodelling, wound healing, fibrosis, and cancer. This review focuses on the developmental heterogeneity of dermal fibroblasts, on their ability to sense changes in biomechanical properties of the surrounding extracellular matrix, and on their role in aging, in skin repair, in pathologic conditions and in tumour development. Moreover, we describe the use of fibroblasts in different models (e.g., in vivo animal models and in vitro systems from 2D to 6D cultures) for tissue bioengineering and the informative potential of high-throughput assays for the study of fibroblasts under different disease contexts for personalized healthcare and regenerative medicine applications.

Utility of fibroblasts derived from broncho-alveolar lavage of patients with idiopathic pulmonary fibrosis or related disorders to develop in vitro models

Broncho-alveolar lavage (BAL) represents a safe tool for the differential diagnosis of various pulmonary fibrotic diseases. Idiopathic pulmonary fibrosis (IPF) belongs to a heterogeneous group of diseases, interstitial lung disease (ILD), presenting a progressive impairment of pulmonary functions. IPF is characterized by the excessive accumulation of extracellular matrix (ECM) in the alveolar parenchyma that may lead to irreversible pulmonary remodeling. Although the exact pathogenetic mechanisms leading to IPF development are still unclear it has been demonstrated that fibroblasts differentiating toward myofibroblasts are the major actors involved in this process. The possibility of obtaining and expanding fibroblasts from the BAL of ILD patients for research purposes has been recently explored. This approach is discussed here as a reliable chance, helpful to advance the scientific community knowledge and to devise two- and three-dimensional (2D/3D) pre-clinical in vitro models of these diseases, further overcoming technical and ethical concerns related to the use of fibroblasts derived from tissue biopsy.

Потенциальные возможности биоинженерных технологий и клеточной имплантации при герниоэндо-протезировании (обзорная статья)

The aim of this review is to determine the most effective way to improve hernioplasty in accordance with the literature data. Materials and methods. The scientific literature was analyzed using PubMed, Scopus, RINC, and Mendeley abstracting software. The review presents data reflecting the trends in the field under consideration over the last decade, according to the authors' opinion. Results. The use of cellular technologies can confirm the position of absorbable hernioprostheses by improving the quality of connective tissue of the postoperative scar and reducing the likelihood of postoperative complications. In the practice of wound treatment, collagen preparations and native fibroblasts are used, the use of which has positive data indicating a high potential of this type of therapy. The application of cell technologies could confirm the position of resorbable hernioprostheses by improving the quality of connective tissue of the postoperative scar and reducing the possibility of postoperative complications. Conclusion. Cellular implantation during hernioendoprosthetics is one of the most promising ways to solve the problem of preventive treatment of postoperative hernias of the anterior abdominal wall. The use of fibroblasts to enhance proliferative activity in the surgical wound will improve the quality characteristics of connective tissue and provide a more favorable long-term effect of the intervention, as well as prevent recurrence of hernias.

Next-generation pacemakers: from electrical devices to biological pacemakers

The invention of an electric pacemaker in the middle of the 20th century led to a revolution in the treatment of cardiac conduction system diseases. The improvement of pacemakers continued. In 1962, the first small series of external pacemakers for percutaneous and direct stimulation was produced in Kaunas. After a while, electric pacemakers became more reliable, smaller and lighter in weight, but the problem of foreign body associated infection and limited service life remained unresolved. Modern high-tech medicine strives to create less invasive electric pacemakers, but nevertheless, biological pacemakers can expand the therapeutic arsenal for the treatment of cardiac patients, being the most physiological for humans. The concept of an artificial biological pacemaker consists of the creation of an organic structure that generates a spontaneous rhythm from the implantation site in the myocardium. Various gene and cellular approaches were used to create biological pacemakers: a functional reorganization approach (use of adenovirus vectors for hyperexpression of genes encoding ion channels in cardiomyocytes); hybrid approach (use of fibroblasts to deliver genes of ion channels that provide heart automation); somatic reprogramming approach (overexpression of the transcription factor TBX18 using adenoviral vectors, which reprograms cardiomyocytes into induced sinoatrial node cells, creating cardiac stimulatory activity); cellular approach (transplantation of stem cells to a specific place in the heart, thereby creating biological stimulation). Modern methods of electrical cardiac stimulation and the developed concepts of the biological pacemaker clearly show the possibility of eliminating current problems associated with the use of an artificial pacemaker by replacing it with a biological one. Each of the approaches (gene, cellular, hybrid-cellular, somatic reprogramming) has its own advantages and disadvantages, which predisposes to further study and improvement in order to introduce a biological pacemaker into clinical practice.

A mitochondria-targeted caffeic acid derivative reverts cellular and mitochondrial defects in human skin fibroblasts from male sporadic Parkinson's disease patients

Parkinson's Disease (PD) is a neurodegenerative disorder affecting more than 10 million people worldwide. Currently, PD has no cure and no early diagnostics methods exist. Mitochondrial dysfunction is presented in the early stages of PD, and it is considered an important pathophysiology component. We have previously developed mitochondria-targeted hydroxycinnamic acid derivatives, presenting antioxidant and iron-chelating properties, and preventing oxidative stress in several biological models of disease. We have also demonstrated that skin fibroblasts from male sporadic PD patients (sPD) presented cellular and mitochondrial alterations, including increased oxidative stress, hyperpolarized and elongated mitochondria and decreased respiration and ATP levels. We also showed that forcing mitochondrial oxidative phosphorylation (OXPHOS) in sPD fibroblasts uncovers metabolic defects that were otherwise hidden. In this work, we tested the hypothesis that a lead mitochondria-targeted hydroxycinnamic acid derivative would revert the phenotype found in skin fibroblasts from sPD patients.Our results demonstrated that treating human skin fibroblasts from sPD patients with non-toxic concentrations of AntiOxCIN4 restored mitochondrial membrane potential and mitochondrial fission, decreased autophagic flux, and enhanced cellular responses to stress by improving the cellular redox state and decreasing reactive oxygen species (ROS) levels. Besides, fibroblasts from sPD patients treated with AntiOxCIN4 showed increased maximal respiration and metabolic activity, converting sPD fibroblasts physiologically more similar to their sex- and age-matched healthy controls. The positive compound effect was reinforced using a supervised machine learning model, confirming that AntiOxCIN4 treatment converted treated fibroblasts from sPD patients closer to the phenotype of control fibroblasts.Our data points out a possible mechanism of AntiOxCIN4 action contributing to a deeper understanding of how the use of mitochondria-targeted antioxidants based on a polyphenol scaffold can be used as potential drug candidates for delaying PD progression, validating the use of fibroblasts from sPD patients with more active OXPHOS as platforms for mitochondria-based drug development.

Validation of reference genes for use in untreated bovine fibroblasts

Proper normalization of RT-qPCR data is pivotal to the interpretation of results and accuracy of scientific conclusions. Though different approaches may be taken, normalization against multiple reference genes is now standard practice. Genes traditionally used and deemed constitutively expressed have demonstrated variability in expression under different experimental conditions, necessitating the proper validation of reference genes prior to utilization. Considering the wide use of fibroblasts in research and scientific applications, it is imperative that suitable reference genes for fibroblasts of different animal origins and conditions be elucidated. Previous studies on bovine fibroblasts have tested limited genes and/or samples. Herein, we present an extensive study investigating the expression stability of 16 candidate reference genes across 7 untreated bovine fibroblast cell lines subjected to controlled conditions. Data were analysed using various statistical tools and algorithms, including geNorm, NormFinder, BestKeeper, and RefFinder. A combined use of GUSB and RPL13A was determined to be the best approach for data normalization in untreated bovine fibroblasts.

The experience of using “ChitoPran” wound coating for treating a patient with combined injury

Background. Resolution of problems related to burn injuries remains relevant and extremely complex in the modern medicine. Biological wound coatings are actively used in the treatment of patients with burn injury. Researchers at the Scientific Research Institute at the Ochapovsky Regional Clinical Hospital No. 1 have been using the biological wound coating ChitoPran since 2017. This coating creates a dry environment in the wound for borderline depth burns and after autoplasty, which contributes to the acceleration of epithelization. For deeper burns after necrectomy, the ChitoPran accelerates the growth of granulation tissues.
 Clinical case. This article presents the clinical case of the successful treatment of a 17-year-old burn patient with a combined injury. The patient presented with a blunt abdominal injury on the background of a deep burn of the anterior abdominal wall. The patient underwent a median laparotomy on the first day and an early necrectomy with primary cutaneous autoplasty with the ChitoPran closing of the wounds on a day later.
 Discussion. The use of the ChitoPran wound coating in combination with skin autoplasty for the early surgical treatment of burns contributes to the acceleration of epithelization of wounds in comparison with stage-based surgical treatment. This coating is hence comparable in terms of cellular epithelization with the use of fibroblasts.
 Conclusions. Creating optimal conditions for epithelization in the wound can prevent the development of purulent complications (such as the failure of skin sutures of the anterior abdominal wall). The use of ChitoPran wound coating showed improved outcomes in the treatment of patients with burn injuries through accelerated restoration of the skin and reduced suppuration in the wound.

Comprehensive Evaluation of the Biological Properties of Surface-Modified Titanium Alloy Implants.

An increasing interest in the fabrication of implants made of titanium and its alloys results from their capacity to be integrated into the bone system. This integration is facilitated by different modifications of the implant surface. Here, we assessed the bioactivity of amorphous titania nanoporous and nanotubular coatings (TNTs), produced by electrochemical oxidation of Ti6Al4V orthopedic implants’ surface. The chemical composition and microstructure of TNT layers was analyzed by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). To increase their antimicrobial activity, TNT coatings were enriched with silver nanoparticles (AgNPs) with the chemical vapor deposition (CVD) method and tested against various bacterial and fungal strains for their ability to form a biofilm. The biointegrity and anti-inflammatory properties of these layers were assessed with the use of fibroblast, osteoblast, and macrophage cell lines. To assess and exclude potential genotoxicity issues of the fabricated systems, a mutation reversal test was performed (Ames Assay MPF, OECD TG 471), showing that none of the TNT coatings released mutagenic substances in long-term incubation experiments. The thorough analysis performed in this study indicates that the TNT5 and TNT5/AgNPs coatings (TNT5—the layer obtained upon applying a 5 V potential) present the most suitable physicochemical and biological properties for their potential use in the fabrication of implants for orthopedics. For this reason, their mechanical properties were measured to obtain full system characteristics.

Transcriptome analysis of fibroblasts from schizophrenia patients reveals differential expression of schizophrenia-related genes

Schizophrenia is a complex neurodevelopmental disorder with high rate of morbidity and mortality. While the heritability rate is high, the precise etiology is still unknown. Although schizophrenia is a central nervous system disorder, studies using peripheral tissues have also been established to search for patient specific biomarkers and to increase understanding of schizophrenia etiology. Among all peripheral tissues, fibroblasts stand out as they are easy to obtain and culture. Furthermore, they keep genetic stability for long period and exhibit molecular similarities to cells from nervous system. Using a unique set of fibroblast samples from a genetically isolated population in northern Sweden, we performed whole transcriptome sequencing to compare differentially expressed genes in seven controls and nine patients. We found differential fibroblast expression between cases and controls for 48 genes, including eight genes previously implicated in schizophrenia or schizophrenia related pathways; HGF, PRRT2, EGR1, EGR3, C11orf87, TLR3, PLEKHH2 and PIK3CD. Weighted gene correlation network analysis identified three differentially co-expressed networks of genes significantly-associated with schizophrenia. All three modules were significantly suppressed in patients compared to control, with one module highly enriched in genes involved in synaptic plasticity, behavior and synaptic transmission. In conclusion, our results support the use of fibroblasts for identification of differentially expressed genes in schizophrenia and highlight dysregulation of synaptic networks as an important mechanism in schizophrenia.

Mitochondrial and autophagic alterations in skin fibroblasts from Parkinson disease patients with Parkin mutations.

PRKN encodes an E3-ubiquitin-ligase involved in multiple cell processes including mitochondrial homeostasis and autophagy. Previous studies reported alterations of mitochondrial function in fibroblasts from patients with PRKN mutation-associated Parkinson’s disease (PRKN-PD) but have been only conducted in glycolytic conditions, potentially masking mitochondrial alterations. Additionally, autophagy flux studies in this cell model are missing.We analyzed mitochondrial function and autophagy in PRKN-PD skin-fibroblasts (n=7) and controls (n=13) in standard (glucose) and mitochondrial-challenging (galactose) conditions.In glucose, PRKN-PD fibroblasts showed preserved mitochondrial bioenergetics with trends to abnormally enhanced mitochondrial respiration that, accompanied by decreased CI, may account for the increased oxidative stress. In galactose, PRKN-PD fibroblasts exhibited decreased basal/maximal respiration vs. controls and reduced mitochondrial CIV and oxidative stress compared to glucose, suggesting an inefficient mitochondrial oxidative capacity to meet an extra metabolic requirement. PRKN-PD fibroblasts presented decreased autophagic flux with reduction of autophagy substrate and autophagosome synthesis in both conditions.The alterations exhibited under neuron-like oxidative environment (galactose), may be relevant to the disease pathogenesis potentially explaining the increased susceptibility of dopaminergic neurons to undergo degeneration. Abnormal PRKN-PD phenotype supports the usefulness of fibroblasts to model disease and the view of PD as a systemic disease where molecular alterations are present in peripheral tissues.

Exosomes from GM-CSF expressing embryonic stem cells are an effective prophylactic vaccine for cancer prevention

ABSTRACTThe antigenic similarity between embryos and tumors has raised the idea of using embryonic material as a preventative vaccine against neoplastic disease. Indeed, we have previously reported that a vaccine comprises allogeneic murine embryonic stem cells (ESCs) and murine fibroblasts expressing GM-CSF (to amplify immune responses) successfully blocks the outgrowth of an implantable cancer (Lewis lung carcinoma; LLC) and lung tumors generated in mice using a combination of a mutagen followed by chronic pulmonary inflammation. However, such a vaccine is obviously impractical for application to humans. The use of fibroblasts to generate GM-CSF is needlessly complicated, and intact whole ESCs carry the hazard of generating embryomas/teratomas. Here, we report the successful application of an alternative prophylactic vaccine comprises exosomes derived from murine ESCs engineered to produce GM-CSF. Vaccination of mice with these exosomes significantly slowed or blocked the outgrowth of implanted LLC while control exosomes lacking GM-CSF were ineffective. Examination of tumor-infiltrating immune cells from mice vaccinated with the GM-CSF-expressing exosomes showed robust tumor-reactive CD8+ T effector responses, Th1 cytokine responses, and higher CD8+ T effector/CD4+CD25+Foxp3+ T regulatory cell ratio in the tumors. We conclude that a similar vaccine derived from GM-CSF- expressing human ESCs can be employed as a preventative vaccine for humans with an increased risk of developing cancer.

Electrospun Schizophyllan/polyvinyl alcohol blend nanofibrous scaffold as potential wound healing

In this study, aqueous Schizophyllan (SPG) (1.5 w/v%) was mixed with aqueous polyvinyl alcohol (PVA) (10 w/v%) at various volume ratios and electrospun to prepare nanofibers. The fiber diameter was decreased by increasing the SPG content. A reliable linear relationship (p < 0.01) was established between the solution properties and fiber diameter. Contiguous, bead-free, and smooth fibers were obtained when the SPG/PVA ratios were 20:80, 30:70, and 40:60. FT-IR spectra, SEM images, tensile testing, swelling ratios, and water contact angle were utilized to characterize this glutaraldehyde (Glu) vapor cross-linked nanofibers in order to analyze the morphology, functional groups, mechanical attributes, hydrophilicity, and humidity of the nanofibers for skin tissue regeneration. Furthermore, the cell culture that was studied by the use of fibroblast (L929) cells showed that these SPG-based nanofibrous scaffolds could generate the improved cell adhesion. In conclusion, it was observed that SPG/PVA nanofiber mat in a volume ratio of 20:80 after 3 day was a suitable material for improving the wound healing, as it could increase cell proliferation and migration that possessed fiber diameter. The characteristics of this nanofiber were 267 nm, contact angle of 47.75°, good swelling behavior (289%), the ultimate strength of 6.513 MPa, Young modulus of 2.665 MPa, and cell viability of 150%.

Cell Type of Origin Dictates the Route to Pluripotency

Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources.

Application of Dermal Fibroblast Cells in Cell Therapy of Wound

Context and Evidence Acquisition: Cell therapy, with autologous dermal fibroblasts, shows remarkable potential and is currently achieved by different methods, such as spraying, grafting, and injecting to treat various types of ulcers, wrinkles, acne scars, and surgical wounds. Results: To prepare fibroblasts for clinical use, some critical points must be taken into consideration: isolation and culture of these cells to produce a homogeneous population of non-contaminated cells, a method for rapid growth, and development of a proportional number of fibroblast cells with proper strength and viability. In addition, an appropriate site for a biopsy should be selected based on indications of treatment and maintenance. Conclusions: Ultimately, the clinical use of fibroblasts in wound healing obviates the need for ready to use reservoir of these cells. In this regard, dermal allografts can be used in both fresh and frozen forms. The fresh type is more desirable due to its high chance of survival. Developing the appropriate preservation methods for allogeneic fibroblasts is the emerging research field of interest. Furthermore, applying biomaterials will increase the efficacy of fibroblasts in wound healing.

Imipramine Inhibits Chikungunya Virus Replication in Human Skin Fibroblasts through Interference with Intracellular Cholesterol Trafficking

Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.

The Use of Fibroblasts for Ameliorating Structural Changes Associated with Skin Aging.

Various causes underlie the structural changes associated with skin aging. To treat these problems, surgical or nonsurgical approaches are available. One of the nonsurgical approaches-regeneration therapy-has become the focus of research and clinical applications. In an earlier clinical study, we injected fibroblasts into the nasolabial region and lips to counter the structural changes associated with skin aging. Assessments performed 12 months after the first injection showed mostly adequate satisfaction scores. However, objective evaluation had not been performed. In the present clinical study, we injected autologous gingival fibroblasts into the nasolabial region and performed objective evaluation using a high-resolution digital camera system 12 months after the first injection. Objective evaluation showed significant improvement in moisture and grain. Subjective evaluation also demonstrated mostly satisfactory results 12 months after the first injection. According to our present results, autologous gingival fibroblast injection might be capable of ameliorating the structural changes associated with skin aging.

Magnesium Modifies the Structural Features of Enzymatically Mineralized Collagen Gels Affecting the Retraction Capabilities of Human Dermal Fibroblasts Embedded within This 3D System

Mineralized collagen gels have been developed as in vitro models to better understand the mechanisms regulating the calcification process and the behavior of a variety of cell types. The vast majority of data are related to stem cells and to osteoblast-like cells, whereas little information is available for dermal fibroblasts, although these cells have been associated with ectopic calcification and consequently to a number of pathological conditions. Therefore, we developed and characterized an enzymatically mineralized collagen gel in which fibroblasts were encapsulated within the 3D structure. MgCl2 was also added during gel polymerization, given its role as (i) modulator of ectopic calcification; (ii) component of biomaterials used for bone replacement; and (iii) constituent of pathological mineral deposits. Results demonstrate that, in a short time, an enzymatically mineralized collagen gel can be prepared in which mineral deposits and viable cells are homogeneously distributed. MgCl2 is present in mineral deposits and significantly affects collagen fibril assembly and organization. Consequently, cell shape and the ability of fibroblasts to retract collagen gels were modified. The development of three-dimensional (3D) mineralized collagen matrices with both different structural features and mineral composition together with the use of fibroblasts, as a prototype of soft connective tissue mesenchymal cells, may pave new ways for the study of ectopic calcification.

PARK2 enhancement is able to compensate mitophagy alterations found in sporadic Alzheimer's disease.

Mitochondrial anomalies have been previously reported in patients' brain and peripheral tissue, suggesting their relevance in sporadic Alzheimer's disease (AD). The present work evaluates mitochondrial function and recycling in human fibroblasts and brain biopsies. Functional studies using patients' skin fibroblasts showed slower mitochondrial membrane potential recovery after a mitochondrial insult together with alterations in lysosomes and autophagy, accompanied by an increase of oxidized and ubiquitinated proteins. Impairment in mitophagy has been proven in these cells due to diminished PARK2 and insufficient vesicle induction, accumulating depolarized mitochondria and PINK1. Augmented Δ1 PINK1 fragment levels suggest an inhibitory effect over PARK2 translocation to the mitochondria, causing the accumulation of activated PINK1. Moreover, the overexpression of PARK2 diminished ubiquitinated proteins accumulation, improves its targeting to mitochondria and potentiates autophagic vesicle synthesis. This allows the reversion of mitophagy failure reflected in the recovery of membrane potential and the decrease of PINK1 and mitochondria accumulation. Sporadic AD fibroblasts exhibited alterations similar to what it could be found in patients' hippocampal samples at early stages of the disease, where there was an accumulation of PINK1 and Δ1 PINK1 together with abnormally increased mitochondrial content. Our findings indicate that mitophagy alterations can be considered a new hallmark of sporadic AD and validate the use of fibroblasts for modelling this pathology.

Mitochondria: Mitochondrial OXPHOS (dys) function ex vivo – The use of primary fibroblasts

Mitochondria are intracellular organelles present in all nucleated cells. They perform a number of vital metabolic processes but their main function is to generate energy in the form of ATP by oxidative phosphorylation (OXPHOS), performed by the mitochondrial respiratory chain. Mitochondrial diseases affecting oxidative phosphorylation are a common group of inherited disorders with variable clinical manifestations. They are caused by mutations either in the mitochondrial or the nuclear genome. In order to study this group of heterogeneous diseases, they are often modeled in animal and microbial systems. However, these are complex, time consuming and unavailable for each specific mutation. Conversely, skin fibroblasts derived from patients provide a feasible alternative. The usefulness of fibroblasts in culture to verify and study the pathomechanism of new mitochondrial diseases and to evaluate the efficacy of individual treatment options is summarized in this review.