The collagenic architecture of human dura mater: Laboratory investigation

Object. Human dura mater is the most external meningeal sheet surrounding the CNS. It provides an efficient protection to intracranial structures and represents the most important site for CSF turnover. Its intrinsic architecture is made up of fibrous tissue including collagenic and elastic fibers that guarantee the maintenance of its biophysical features. The recent technical advances in the repair of dural defects have allowed for the creation of many synthetic and biological grafts. However, no detailed studies on the 3D microscopic disposition of collagenic fibers in dura mater are available. The authors report on the collagenic 3D architecture of normal dura mater highlighting the orientation, disposition in 3 dimensions, and shape of the collagen fibers with respect to the observed layer. Methods. Thirty-two dura mater specimens were collected during cranial decompressive surgical procedures, fixed in 2.5% Karnovsky solution, and digested in 1 N NaOH solution. After a routine procedure, the specimens were observed using a scanning electron microscope. Results. The authors distinguished the following 5 layers in the fibrous dura mater of varying thicknesses, orientation, and structures: bone surface, external median, vascular, internal median, and arachnoid layers. Conclusions. The description of the ultrastructural 3D organization of the different layers of dura mater will give us more information for the creation of synthetic grafts that are as similar as possible to normal dura mater. This description will be also related to the study of the neoplastic invasion

Ultrastructural localization of tyrosine hydroxylase in human peripheral blood mononuclear cells: effect of stimulation with phytohaemagglutinin

Using immunocytochemistry coupled to fluorescence and electron microscopy, we investigated the expression and ultrastructural localization of tyrosine hydroxylase (TH, EC 1.14.16.2), the rate-limiting enzyme in the biosynthesis of catecholamines, in human peripheral blood mononuclear cells (PBMCs), with PC12 cells as positive controls. In unstimulated PBMCs, TH-specific immunoreactivity was localized to the plasma membrane. However, after stimulation with the polyclonal mitogen phytohaemagglutinin (PHA), TH immunoreactivity was almost completely localized to electron-dense cytoplasmic granules, which resembled those found in PC12. TH-positive granules, however, were larger (300-500 nm) than in PC12 cells (100-200 nm). Flow cytometry analysis of TH expression showed about 46-50% positive cells in unstimulated PBMCs and in PHA-stimulated PBMCs in the G0/G1 phase of the cell cycle, but more than 80% positive cells in PHA-stimulated PBMCs in the S+G2/M phase. In agreement with previous observations, PHA stimulation also induced de novo expression of TH mRNA as well as increased intracellular catecholamine content, suggesting the occurrence of TH upregulation at the level of both gene expression and enzyme activity. The ultrastructural localization of TH in human PBMCs seems therefore regulated by cell stimulation and related to the functional activity of the enzyme

Self-Healing Collagen-Based Hydrogel for Brain Injury Therapy

Hydrogels derived from biopolymers, also called biohydrogels, have shown potential for brain injury therapy due to their tunable physical, chemical, and biological properties. Among different biohydrogels, those made from collagen type I are very promising candidates for the reparation of nervous tissues due to its biocompatibility, noncytotoxic properties, injectability, and self-healing ability. Moreover, although collagen does not naturally occur in the brain, it has been demonstrated that collagen type I, which resides in the basal lamina of the subventricular zone in adults, supports neural cell attachment, axonal growth, and cell proliferation due to its intrinsic content of specific cell-signaling domains. This chapter summarizes the most relevant results obtained from both in vitro and in vivo studies using self-healing biohydrogels based on collagen type I as key component in the field of neuroregeneration.University of RegensburgUniversidad de La LagunaMinisterio de Ciencia, Innovación y Universidade

Impianto di condrociti autologhi: analisi ultrastrutturale, biochimica e dell\u2019espressione genica.

L'impianto di condrociti autologhi su membrana collagenica (MACI) \ue8 una tecnica di ingegneria tissutale per il trattamento delle lesioni cartilaginee articolari a tutto spessore. Tale metodica prevede la semina di condrociti coltivati su una membrana di collagene tipo I/III, la quale \ue8 impiantata nel difetto condrale utilizzando esclusivamente colla di fibrina. Abbiamo analizzato l\u2019evoluzione strutturale e molecolare del MACI dopo l\u2019impianto. L\u2019analisi morfologica \ue8 stata eseguita su 3 campioni bioptici, prelevati da 3 differenti pazienti a 2 anni di follow-up, utilizzando metodiche di microscopia luce, immunoistochimica (collagene tipo I e II), SEM e TEM. Per la valutazione biochimica \ue8 stata utilizzata la Flurophore Assisted Carbohydrate Electrophoresis (FACE), per l\u2019analisi dell\u2019espressione genica la Quantitative Real Time PCR (QRT-PCR). L\u2019analisi istologica ha mostrato la formazione di un tessuto ialino in tutti i casi, con presenza di collagene tipo II, condroitinsolfato e la formazione del tidemark. In un solo campione si \ue8 notato la concomitante positivit\ue0 per il collagene tipo I limitatamente allo strato superficiale. Sono state evidenziate all\u2019esame ultrastrutturale la riorganizzazione del network fibrillare collagenico e la organizzazione in cluster degli elementi cellulari. La FACE ha dimostrato la presenza di glicosaminoglicani con un pattern simile ad un tessuto cartilagineo ialino. La QRT-PCR ha registrato l\u2019espressione del gene collagene tipo II in tutti i campioni. In un solo caso vi era la concomitante espressione del gene collagene tipo I. Lo studio dimostra che la membrana di collagene tipo I/III \ue8 un supporto ideale per la crescita di condroblasti i quali, una volta impiantati nel difetto condrale, formano un tessuto simile alla cartilagine ialina