Dual nature of mouse periderm structure, function and fate.

The periderm is the outer layer of embryonic skin, which is essential for the development of the epidermis and the establishment of its barrier function. In humans, the periderm is a monolayer and is shed prenatally. The structure and fate of the mouse periderm remain puzzling. Using electron microscopy of mouse skin between the embryonic day 14.5 and the postnatal Day 2, we have assessed the periderm structure and its shedding patterns. In contrast to human periderm, the mouse periderm appears to be bilayered, comprising a presumably absorptive outer periderm with numerous microvilli and an inner periderm packed with specific (glycogen-containing?) granules. The desmosomes between the inner periderm and the underlying epidermis are scarce, and they are totally absent between the inner and the outer periderm. The inner and outer periderms in mice are shed at different stages of development: the outer periderm is shed in utero in conjunction with barrier acquisition (E16-18), while the inner periderm is retained postnatally. We assume that the bilayered structure of the mouse periderm and the postnatal retention of its inner layer represent the evolutionary adaptations in ancestral rodents which helps altricial newborns of their extant descendants to cope with acute dehydration right after birth and/or provides a substrate for proper bacterial colonization of newborn skin.

Mechanisms driving the initiation and direction of endothelial sprouting in organotypic co-culture of aorta and spinal cord tissues.

The resumption of blood supply in spinal cord (SC) after injury is a prerequisite of its recovery. To expose the mechanisms of damaged SC revascularization we have used an organotypic SC/aortic fragments (AF) co-culture where, as we showed previously, damaged SC tissue induces AF cell sprouting but repels them away. Supplementation of culture medium with exogenous VEGF-A165 redirects the migrating aortic endothelial cells towards SC tissue. This effect and the pattern of sFlt1 expression (a soluble form of VEGFR1) suggest that the low level of SC-secreted VEGF and the presence of sFlt1 in SC slices together prevent the migration of aortic CD31+ cells to the SC in the absence of exogenous VEGF. VEGF-A165 supplementation sequesters this inhibitory activity of sFlt1 by direct binding thus allowing CD31+ cell migration in to SC tissue. Proteome analysis has shown that migration/proliferation of CD31+ and αSMA+ aortic cells in neuronal culture medium used in our SC/AF model (which obstruct sprouting by itself) was resumed by combined action of several pro- (aFGF, bFGF, Osteopontin, TF, IGFBP2, SDF1) and anti-angiogenic (Endostatin/Collagen18) factors. The mutual influence of AF and SC tissues is a key factor balancing these factors and thus driving endothelial sprouting in SC injury zone.

Spinal cord tissue affects sprouting from aortic fragments in ex vivo co-culture.

It is a well-known fact, that there is a close interconnection between vascular and neural structures in both embryonic development and postnatal life. Different models have been employed to dissect the mechanisms of these interactions, ranging from in vitro systems (e.g., co-culture of neural and endothelial cells) to in vivo imaging of central neural system recovery in laboratory animals after artificially induced trauma. Nevertheless, most of these models have serious limitations. Here, we describe an ex vivo model, representing an organotypic co-culture of aortic fragments (AF) with longitudinal slices of mouse neonatal spinal cord (SC) or dorsal root ganglia (DRG). The samples were co-cultured in a medium adapted for SC tissue and lacking any pro-angiogenic or neurotrophic growth factors. It was found, that cultivation of AFs in the SC injury zone (transversal dissection of a SC slice) resulted in the initiation of active aortic sprouting. Remarkably, the endothelial cells exiting the AFs never invaded the SC tissue, concentrating in a nearby area (negative taxis). In contrast, the DRGs, while also promoting the sprouting, were a target of active endothelial CD31+ cell invasion (positive taxis). Thus, the tissues of both central and peripheral nervous systems have a prominent positive effect on aortic sprouting, while the vector of endothelial cell expansion is strictly nervous-tissue-type dependent. The ex vivo AF co-culture with SC or DRG appeared to be a useful and promising model for a further endeavor into the mechanisms driving the complex interactions between neural and endothelial tissues.

Primary culture of mouse embryonic spinal cord neurons: cell composition and suitability for axonal regeneration studies.

Objective: Primary culture is an effective experimental model to study molecular mechanisms that drive axonal regeneration after central nervous system injury. However, the culture of spinal cord (SC) cells remains poorly characterized. Here, we have analyzed the cell composition of a primary SC culture during its maturation. Methods: Primary cell culture was prepared from mouse embryo spinal cords. After 2, 7, and 14 days of cultivation, the cells were fixed and stained with antibodies against ß3-tubulin, nestin, crmp1, SMI-32, DCC or GFAP. We counted percentage of cells positive for the mentioned markers and measured the length of cell processes. Results: We found that ß3-tubulin and nestin were both expressed at day 2 of culture in vitro. Surprisingly (given the use of differentiation-supporting culture medium), the number of nestin+ cells has significantly increased during the first week of cultivation. The GFAP+ cells appeared only at the seventh day in vitro, and their fraction increased during the following cultivation. At 14 day in vitro, SC culture contained cells that expressed the markers typical of commissural and motor neurons. At this age, the neurons had the ability to repair injured neurites after mechanical damage. Conclusion: Primary culture of SC cells is a dynamically developing cell population that contains all main types of SC cells and is capable of self-repair. Therefore, the culture of mouse embryonic SC cells represents an adequate experimental model for studying cellular and molecular processes taking place in SC neurons after axonal damage in the absence of external inhibitors.

Retinoic acid co-treatment aggravates severity of dioxin-induced skin lesions in hairless mice via induction of inflammatory response.

Exposure to toxic halogenated polyaromatic hydrocarbons, of which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent, induces diverse skin pathologies in humans, including chloracne, hyperkeratosis, hamartomas, etc. While the toxic effects of TCDD have been extensively studied, effective approaches to their treatment are still lacking. Retinoids are commonly used in therapy of acneiform skin diseases. In vitro, retinoids elicit antagonistic effects on keratinocyte differentiation and proliferation, as compared to TCDD, suggesting their potential in treatment of TCDD-induced skin lesions. Nevertheless, the modulation of TCDD activity in skin by retinoids in vivo was never reported. We have used N-TERT keratinocyte cell line and hairless (hr) mice to determine if retinoic acid (RA) can lessen or reverse TCDD-induced effects in vitro and in vivo. RA co-treatment suppressed TCDD-induced changes in the expression of differentiation-associated genes and N-TERT keratinocyte viability in vitro. However, in hairless mice (in vivo), RA/TCDD co-treatment produced more severe effects, than treatment with either of the two compounds individually. RA/TCDD co-application to mouse skin strongly stimulated keratinocyte proliferation, resulting in dramatic epidermal hyperplasia. It has also led to massive immune cell infiltration into the dermis, and increased mRNA expression of inflammation markers, including IL1ß, IL6 and S100A7. Thus, retinoids not only appeared ineffective in treatment of TCDD-induced skin lesions in hairless mice, but also resulted in their exaggeration. These in vivo results question previous cell culture-based claims that RA may reduce TCDD-induced skin effects and caution against the reliance on in vitro data in TCDD toxicology research.

Functional anatomy of the hair follicle: The Secondary Hair Germ.

The secondary hair germ (SHG)-a transitory structure in the lower portion of the mouse telogen hair follicle (HF)-is directly involved in anagen induction and eventual HF regrowth. Some crucial aspects of SHG functioning and ontogenetic relations with other HF parts, however, remain undefined. According to recent evidence (in contrast to previous bulge-centric views), the SHG is the primary target of anagen-inducing signalling and a source of both the outer root sheath (ORS) and ascending HF layers during the initial (morphogenetic) anagen subphase. The SHG is comprised of two functionally distinct cell populations. Its lower portion (originating from lower HF cells that survived catagen) forms all ascending HF layers, while the upper SHG (formed by bulge-derived cells) builds up the ORS. The predetermination of SHG cells to a specific morphogenetic fate contradicts their attribution to the "stem cell" category and supports SHG designation as a "germinative" or a "founder" cell population. The mechanisms of this predetermination driving transition of the SHG from "refractory" to the "competent" state during the telogen remain unknown. Functionally, the SHG serves as a barrier, protecting the quiescent bulge stem cell niche from the extensive follicular papilla/SHG signalling milieu. The formation of the SHG is a prerequisite for efficient "precommitment" of these cells and provides for easier sensing and a faster response to anagen-inducing signals. In general, the formation of the SHG is an evolutionary adaptation, which allowed the ancestors of modern Muridae to acquire a specific, highly synchronized pattern of hair cycling.

ARNT controls the expression of epidermal differentiation genes through HDAC- and EGFR-dependent pathways.

Previously we showed that spatial and developmental modulation of ARNT (HIF1ß) expression in mouse epidermis is essential for maintenance of keratinocyte differentiation, proper formation of the barrier and normal desquamation. Here, using lentiviral suppression or induction of ARNT in TERT-immortalized (N-TERT) and HaCaT cells we assessed the nature and mechanisms of ARNT involvement in control of differentiation in human epidermal keratinocytes. ARNT depletion did not affect the levels of basal keratins K5 and K14, but significantly induced expression of several key differentiation markers (an effect abolished by EGF supplementation). Furthermore, ARNT deficiency resulted in the downregulation of amphiregulin (AREG) - the most highly expressed EGFR ligand in human keratinocytes - whereas upregulation of ARNT showed the opposite. In ARNT-deficient monolayer cultures and 3D epidermal equivalents, the downregulation of AREG was concurrent with a decline of EGFR and ERK1/2 phosphorylation. TSA, a potent suppressor of HDAC activity, abolished the effects of ARNT deficiency, implying a role for HDACs in ARNT-dependent modulation of the AREG-EGFR pathway and downstream epidermal genes. Total HDAC activity was significantly increased in ARNT-depleted cells and decreased with ARNT overexpression. ARNT-dependent shifts in HDAC activity were specifically attributed to significant changes in the levels of HDAC1, HDAC2 and HDAC3 proteins (but not mRNA) in both monolayer and 3D cultures. Collectively, our results suggest that ARNT controls AREG expression and the downstream EGFR-ERK pathway in keratinocytes, at least in part, by modulating HDAC activity. This novel regulatory pathway targeting advanced stages of epidermal differentiation might have important implications for skin pathology such as psoriasis, atopic dermatitis and cancer.

Fibroblasts and polymer composition are essential for bioengineering of airway epithelium on nonwoven scaffolds.

To make tissue engineering a truly effective tool, it is necessary to understand how the patterns of specific tissue development are modulated by and depend on the artificial environment. Even the most advanced approaches still do not fully meet the requirements of practical engineering of tracheobronchial epithelium. This study aimed to test the ability of the synthetic and natural nonwoven scaffolds to support the formation of morphological sound airway epithelium including the basement membrane (BM). We also sought to identify the potential role of fibroblasts in this process. Our results showed that nonwoven scaffolds are generally suitable for producing well-differentiated tracheobronchial epithelium (with cilia and goblet cells), while the structure and functionality of the equivalents appeared to be highly dependent on the composition of the scaffolds. Unlike natural scaffolds, synthetic ones supported the formation of the epithelium only when epithelial cells were cocultured with fibroblasts. Fibroblasts also appeared to be obligatory for basal lamina formation, regardless of the type of the nonwoven material used. However, even in the presence of fibroblasts, the synthetic scaffolds were unable to support the formation of the epithelium and of the BM (in particular, basal lamina) as effectively as the natural scaffolds did.

Effect of collagen denaturation degree on mechanical properties and biological activity of nanofibrous scaffolds.

Further progress in regenerative medicine and bioengineering highly depends on the development of 3D polymeric scaffolds with active biological properties. The most attention is paid to natural extracellular matrix components, primarily collagen. Herein, nonwoven nanofiber materials with various degrees of collagen denaturation and fiber diameters 250-500 nm were produced by electrospinning, stabilized by genipin, and characterized in detail. Collagen denaturation has been confirmed using DSC and FTIR analysis. The comparative study of collagen and gelatin nonwoven materials (NWM) revealed only minor differences in their biocompatibility with skin fibroblasts and keratinocytes in vitro. In long-term subcutaneous implantation study, the inflammation was less evident on collagen than on gelatin NWM. Remarkably, the pronounced calcification was revealed in the collagen NWM only. The results obtained can be useful in terms of improving the electrospinning technology of collagen from aqueous solutions, as well as emphasize the importance of long-term study to ensure proper implementation of the material, taking into account the ability of collagen to provoke calcification.

Hypoxia-mediated control of HIF/ARNT machinery in epidermal keratinocytes.

Transcriptional activity of hypoxia-induced factor 1 (HIF1) - a heterodimer of HIF1α and ARNT (HIF1ß) - is essential for cellular adaptation to environmental stress and plays an important role in skin development, wound healing, tumorigenesis and barrier function. Using primary mouse and human epidermal keratinocytes at ambient or hypoxic (1% O(2)) conditions we studied effects of hypoxia upon HIF protein expression. Significant nuclear levels of ARNT and HIF1α along with high HIF1 activity in normoxic keratinocytes suggest an as yet uncharacterised oxygen-independent role for HIF pathway in the epidermis. Acute hypoxia results in an instant but transient increase of HIF1α protein accompanied by a gradual decrease in its mRNA, while ARNT expression remains unchanged. In prolonged (chronic) hypoxia both HIF1α and Arnt are downregulated along with decline of HIF1 activity. However, expression of classical HIF1 targets such as Selenbp1 and Vegfa remains high. Thus, keratinocytes respond to acute hypoxia with immediate block of HIF1α protein degradation and concomitant increase of HIF activity, while under chronic hypoxia pro-angiogenic signalling is maintained through HIF1-independent pathway(s). Decline of HIF1α during chronic exposure is controlled at both mRNA and protein levels, while Arnt is downregulated post-translationally. Distinct transcription levels of Hif1α and Hif3α splice variants under normoxia and their differential response to hypoxia suggest functional diversity of Hif-α isoforms and highlight the complexity of HIF machinery control in epidermal keratinocytes.

Loss of Arnt (Hif1ß) in mouse epidermis triggers dermal angiogenesis, blood vessel dilation and clotting defects.

Targeted ablation of Aryl hydrocarbon receptor nuclear translocator (Arnt) in the mouse epidermis results in severe abnormalities in dermal vasculature reminiscent of petechia induced in human skin by anticoagulants or certain genetic disorders. Lack of Arnt leads to downregulation of Egln3/Phd3 hydroxylase and concomitant hypoxia-independent stabilization of hypoxia-induced factor 1α (Hif1α) along with compensatory induction of Arnt2. Ectopic induction of Arnt2 results in its heterodimerization with stabilized Hif1α and is associated with activation of genes coding for secreted proteins implicated in control of angiogenesis, coagulation, vasodilation and blood vessel permeability such as S100a8/S100a9, S100a10, Serpine1, Defb3, Socs3, Cxcl1 and Thbd. Since ARNT and ARNT2 heterodimers with HIF1α are known to have different (yet overlapping) downstream targets our findings suggest that loss of Arnt in the epidermis activates an aberrant paracrine regulatory pathway responsible for dermal vascular phenotype in K14-Arnt KO mice. This assumption is supported by a significant decline of von Willebrand factor in dermal vasculature of these mice where Arnt level remains normal. Given the essential role of ARNT in the adaptive response to environmental stress and striking similarity between skin vascular phenotype in K14-Arnt KO mice and specific vascular features of tumour stroma and psoriatic skin, we believe that further characterization of Arnt-dependent epidermal-dermal signalling may provide insight into the role of macro- and micro-environmental factors in control of skin vasculature and in pathogenesis of environmentally modulated skin disorders.

Nonwoven spidroin materials as scaffolds for ex vivo cultivation of aortic fragments and dorsal root ganglia.

Recombinant spidroins (RS; the analogues of silk proteins of spider's web) have multiple properties beneficial for bioengineering, including their suitability for electrospinning and thus, for production of materials with oriented fibers. This makes RS-based matrices potentially effective in stimulating regeneration of peripheral nerves. The restoration of injured nerves also depends on prompt regrowth of blood vessels. Therefore, prospective scaffold materials for neuro-regenerative therapy should positively affect both the nerves and the blood vessels. Currently, the experimental models suitable for culturing and quantitative assessment of the vascular and neuronal cells on the same material are lacking. Here, we assessed the suitability of electrospun RS-based matrices for cultivation of the mouse aorta and dorsal root ganglia (DRG) explants. We also quantified the effects of matrix topography upon both types of tissues. The RS-based materials have effectively supported aortic explants survival and sprouting. The cumulative length of endothelial sprouts on rS1/9-coated inserts was significantly higher as compared to type I collagen coatings, suggesting stimulatory effects on angiogenesis in vitro. In contrast to matrices with random fibers, on matrices with parallel fibers the migration of both smooth muscle and endothelial cells was highly oriented. Furthermore, alignment of RS fibers effectively directs the growth of axons and the migration of Schwann cells from DRGs. Thus, the electrospun RS matrices are highly suitable to culture both, the DRGs and aortic explants and to study the effects of matrix topography on cell migration. This model has a high potential for further endeavor into interactions of nerve and vascular cells and tissues.

The reduction of water-soluble tetrazolium salt reagent on the plasma membrane of epidermal keratinocytes is oxygen dependent.

The water-soluble tetrazolium salt (WST-1) assay is frequently used to assess cell proliferation. However, our study showed that in normal and cancerous keratinocytes, this assay is more responsive to changes in oxygenation than to rates of cell growth. Stimulation of keratinocyte proliferation by low Ca(2+) and suppression of proliferation by nocodazole resulted in modest changes in WST-1 readings, whereas gradually reducing the level of oxygen in the cellular environment from ambient (21%) to near anoxic (0.1%) revealed a very strong negative correlation between cell oxygenation and WST-1 reagent reduction. In contrast, the very similar MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay, which uses a different tetrazolium salt, showed no sensitivity to the level of oxygen. Unlike MTT, WST-1 reagent is reduced extracellularly through trans-plasma membrane transport (tPMET), thereby suggesting that tPMET is oxygen dependent. We propose that the WST-1 assay can be developed into a sensitive quantitative method to evaluate cell oxygenation in vitro and used to study the role of hypoxia and tPMET in homeostasis and disease (e.g., cancer). At the same time, WST-1 assay should be used cautiously to assess cell viability or proliferation because readings can be affected by certain extrinsic (low atmospheric oxygen or high density culture) or intrinsic (defects in oxygen-sensing pathways) factors.

Epidermal expression of the truncated prelamin A causing Hutchinson-Gilford progeria syndrome: effects on keratinocytes, hair and skin.

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by point mutation in LMNA encoding A-type nuclear lamins. The mutations in LMNA activate a cryptic splice donor site, resulting in expression of a truncated, prenylated prelamin A called progerin. Expression of progerin leads to alterations in nuclear morphology, which may underlie pathology in HGPS. We generated transgenic mice expressing progerin in epidermis under control of a keratin 14 promoter. The mice had severe abnormalities in morphology of skin keratinocyte nuclei, including nuclear envelope lobulation and decreased nuclear circularity not present in transgenic mice expressing wild-type human lamin A. Primary keratinocytes isolated from these mice had a higher frequency of nuclei with abnormal shape compared to those from transgenic mice expressing wild-type human lamin A. Treatment with a farnesyltransferase inhibitor significantly improved nuclear shape abnormalities and induced the formation of intranuclear foci in the primary keratinocytes expressing progerin. Similarly, spontaneous immortalization of progerin-expressing cultured keratinocytes selected for cells with normal nuclear morphology. Despite morphological alterations in keratinocyte nuclei, mice expressing progerin in epidermis had normal hair grown and wound healing. Hair and skin thickness were normal even after crossing to Lmna null mice to reduce or eliminate expression of normal A-type lamins. Although progerin induces significant alterations in keratinocyte nuclear morphology that are reversed by inhibition of farnesyltransferasae, epidermal expression does not lead to alopecia or other skin abnormalities typically seen in human subjects with HGPS.

Sodium alginate-based composites as a collagen substitute for skin bioengineering.

The skin is a combination of two different types of tissue-epithelial and connective (mesenchymal). The outer protective layer of the skin, the epidermis, consists of multiple layers of keratinocytes residing on the basement membrane that separates them from the underlying dermis, which consists of a well-vascularized fibrous extracellular matrix seeded mainly by fibroblasts and mesenchymal stromal cells. These skin features suggest that the development of a fibroblast-friendly porous scaffold covered with a flat dense sheath mimicking the basement membrane, and sufficient to support keratinocyte attachment, would be a reasonable approach in the generation of clinically-relevant skin substitutes useful for reconstructive dermatology and burn treatment. Therefore, we developed a procedure to obtain biocompatible composite bilayer scaffolds comprising a spongy dermis-like body (supporting vascularization and appropriate fibroblast and multipotent stromal cell activity) fused with a film-like cover (supporting keratinocyte attachment, growth and differentiation). The sodium alginate (SA), an algae-derived biopolymer, has been used as a base component for these scaffolds while collagen (CL) and fibrinogen (FG) were used as minor additives in variable concentrations. The slow rates of composite SA-based scaffold biodegradation were achieved by using Ba2+ as cross-linking cations. By manipulating the SA/CL/FG ratio we managed to obtain sponge scaffolds with highly interconnected porous structures, with an average pore size ranging from 60 to 300 µm, and sufficient tensile strength (3.12-5.26 MPa). The scaffolds biocompatibility with the major human skin cell types was confirmed by seeding the scaffold sponge compartment with primary skin fibroblasts and subcutaneous adipose-derived stromal cells while the film side biocompatibility was tested using primary human keratinocytes. The obtained results have shown that bilayer alginate-based scaffolds have biological and mechanical properties comparable with CL scaffolds but surpass them in cost efficiency and vascularization ability in the subcutaneous implantation model in laboratory mice.

Endoscopic Endonasal Transclival Approach to Tumors of the Clivus and Anterior Region of the Posterior Cranial Fossa (Results of Surgical Treatment of 136 Patients).

INTRODUCTION: With the introduction into the neurosurgical practice of minimally invasive methods using endoscopic techniques, it became possible to effectively remove hard-to-reach tumors, including central tumors of the anterior region of the posterior cranial fossa. OBJECTIVE: To analyze the results of surgical treatment of patients with various centrally located tumors of the base of the skull that extend into the anterior region of the posterior cranial fossa using the endoscopic endonasal transclival approach. METHODS: The personal surgical experience of the first author is 136 patients with various tumors (e.g., chordomas, meningiomas, pituitary adenomas, and fibrous dysplasia). RESULTS: Radicality of tumor removal was total 61.03%; subtotal 25%; partial 13.23%; and insufficient removal 0.74%. Postoperative cerebrospinal fluid leaks occurred in 9 cases (6.62%) and meningitis in 13 cases (9.56%). In 2 cases, surgical treatment had a lethal outcome (1.47%). CONCLUSIONS: The endoscopic endonasal transclival approach can be used to obtain access to centrally located skull base tumors. This approach allows for a radical and low-risk removal of various skull base tumors of central localization that, until recently, were considered to be almost inoperable.

Experimental animal model for assessment of tracheal epithelium regeneration.

OBJECTIVES/HYPOTHESIS: To develop an experimental model in rabbits for assessment of tracheal epithelium regeneration through application of either natural or artificial polymer scaffolds. STUDY DESIGN: First, we identified the size of full-thickness mucosal defect, which does not allow self-healing (a "critical defect"), thus representing an adequate experimental model for regenerative therapy of tracheal epithelium damage. Then, two methods of polymer scaffold fixation at the site of the epithelium defect were compared: suturing and fixation with a stent. This was done through: 1) formation of a full-thickness anterolateral mucosal defect by tracheal mucosa excision; and 2) fixation of the scaffold at the site of the tracheal epithelium defect using sutures (through a tracheal wall "window") or a vascular stent (through a small tracheal incision). RESULTS: The dimension of a critical anterolateral mucosal defect of the trachea for rabbits was found to be 1.5 cm in length and more than 50% of the tracheal circumference. Fixation of the scaffold with a stent proved to be more efficient due to a uniform distribution of the pressure over the entire surface of the scaffold, whereas the suturing of the scaffold provided unsatisfactory results. In addition, fixation of the scaffold by suturing required formation of a large "window" in the tracheal wall. Thus, using the stent appeared to be technically less complicated and much less traumatic as compared to suturing. CONCLUSION: We present an experimental in vivo animal model of tracheal epithelium injury and recovery. It can be effectively used with certain further modifications as a basis for routine testing of bioengineered constructs. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E213-E219, 2019.

Non-woven bilayered biodegradable chitosan-gelatin-polylactide scaffold for bioengineering of tracheal epithelium.

OBJECTIVES: The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non-woven material in order to obtain a well-differentiated human airway epithelium. MATERIALS AND METHODS: The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy. RESULTS: With the use of a copolymer of chitosan-gelatin-poly-l-lactide, a bilayered non-woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed. CONCLUSIONS: The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well-differentiated equivalent of respiratory epithelium on non-woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co-culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.

Endoscopic endonasal transclival removal of tumors of the clivus and anterior region of the posterior cranial fossa (results of surgical treatment of 140 patients).

BACKGROUND: Until recently, tumors of the clivus and the anterior region of the posterior cranial fossa were considered extremely difficult to access and often inoperable using standard transcranial approaches. With the introduction into the neurosurgical practice of minimally invasive methods utilizing endoscopic techniques, it became possible to effectively remove hard-to-reach tumors, including central tumors of the anterior region of the posterior cranial fossa. METHODS: From 2008 to the present time, the inpatient institution has operated on 140 patients with various tumors of the base of the skull, localized to the clivus and anterior region of the posterior cranial fossa (65 men and 75 women). The age of patients ranged from 3 to 74 years. Tumor distribution according to the histopathological features was as follows: chordomas, 103 (73.57%); meningiomas, 12 (8.57%); pituitary adenomas, 9 (6.43%); fibrous dysplasia, 4 (2.86%); cholesteatoma, 3 (2.14%); craniopharyngiomas, 2 (1.43%); plasmacytomas, 2 (1.43%); and other tumors (giant cell tumor, neurohypophyseal glioma, osteoma, carcinoid, chondroma), 5 (3.57%). The tumors had the following size distribution: giant (more than 60 mm), 35 (25%); large (35-59 mm), 83 (59.3%); medium (21-35 mm), 21 (15%); and small (less than 20 mm), 1 (0.7%). In 11 cases, intraoperative monitoring of the cranial nerves was performed (21 cranial nerves were identified). RESULTS: Upper, middle, and lower transclival approaches provide access to the anterior surface of the upper, middle, and lower neurovascular complexes of the posterior cranial fossa. The chordoma cases were distributed as follows according to extent of removal: total removal, 68 (66.02%); subtotal removal, 25 (24.27%); and partial removal, 10 (9.71%). The adenomas of the pituitary gland were removed totally in 6 cases, subtotally in 1 case and partially in 2 cases. The meningiomas were removed totally in 1 case, subtotally in 5 cases, and partially in 5 cases, with less than 50% of the tumor removed in 1 case. Other tumors (cholesteatoma, craniopharyngioma, fibrous dysplasia, giant cell tumor, glioma of the neurohypophysis, osteoma, plasmacytoma, carcinoid, and chondroma) were removed totally in 9 cases and subtotally in 7 cases. Postoperative CSF leaks occurred in 9 cases (6.43%) and meningitis in 13 cases (9.29%). Oculomotor disorders developed in 19 patients (13.57%), 12 of which regressed during the period from 4 to 38 days after surgery, and 7 of which were permanent. In 2 cases, surgical treatment had a lethal outcome (1.43%). CONCLUSION: The endoscopic endonasal transclival approach can be used to obtain access to the centrally located tumors of the posterior cranial fossa. It is an alternative to transcranial approaches in the surgical treatment of tumors of the clivus. The results of using this approach are comparable with the results of transcranial and transfacial approaches and, in some cases, surpass them in effectiveness. The extended endoscopic endonasal posterior (transclival) approach, considering its minimally invasive nature, allows fora radical and low-risk (in terms of postoperative complications and lethality) removal of various skull base tumors of central localization with the involvement and without the involvement of the clivus, which, until recently, were considered to be almost inoperable.

Endoscopic Endonasal Transclival Approach to Tumors of the Clivus and Anterior Region of the Posterior Cranial Fossa: An Anatomic Study.

OBJECTIVE: To present the basic topographic and anatomic features of the clivus and adjacent structures with an objective of possible improvements and optimization of the extended endoscopic endonasal posterior (transclival) approach when removing tumors of the clivus and anterior regions of the posterior cranial fossa. MATERIALS AND METHODS: A craniometric study was conducted on 125 human skulls. A topographic anatomic study was conducted on 25 cadaver head specimens with arterial and venous beds stained with colored silicone, according to the method developed by us, to visualize its features and individual variability. RESULTS: The most important anatomic features of the external and internal regions of the clivus and the adjacent neural and vascular structures were analyzed. An accessible zone for the most effective transclival approach to the posterior cranial fossa is also specified. CONCLUSION: The endoscopic endonasal transclival approach can be used to obtain access to centrally located tumors of the posterior cranial fossa. It is an alternative to transcranial approaches in the surgical treatment of tumors of the clivus.