Martin Witt

Synergistic Effect of Titanium Alloy and Collagen Type I on Cell Adhesion, Proliferation and Differentiation of Osteoblast-Like Cells

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors.

Biopsies of olfactory epithelium in patients with Parkinson's disease.

Parkinsons disease (PD) is a neurodegenerative disorder involving several neuronal systems. Impaired olfactory function may constitute one of the earliest symptoms of PD. However, it is still unclear to what degree changes of the olfactory epithelium may contribute to dysosmia and if these changes are different from those of other hyposmic or anosmic patients. This study aimed to investigate the hypothesis that olfactory loss in PD is a consequence of specific PD‐related damage of olfactory epithelium. Biopsies of 7 patients diagnosed with PD were taken. Six patients with PD were hyposmic, one anosmic. As non‐PD controls served 9 patients with hyposmia, 9 with anosmia, and 7 normosmic individuals. Further, nasal mucosa of 4 postmortem individuals was investigated. Immunohistochemical examinations were performed with antibodies against olfactory marker protein (OMP), protein gene product 9.5 (PGP 9.5), beta‐tubulin, (BT), proliferation‐associated antigen (Ki 67), the stem cell marker nestin, cytokeratin, p75NGFr, and α‐synuclein. Most of the biopsy specimens exhibited irregular areas of olfactory‐like, dysplastic epithelium positive for either PGP 9.5 or BT, but negative for OMP. No major histochemical differences in either the expression or distribution of these proteins were observed in the olfactory epithelium of patients with PD compared with controls. Reverse transcription PCR (RT‐PCR) data indicated mRNA for OMP in almost all subjects, independently of their olfactory performance. These data support the idea that olfactory loss in Parkinsons disease is not a consequence of damage to the olfactory epithelium but rather results from distinct central‐nervous abnormalities.

Immunohistochemical detection of human skin nerve fibers

The autonomic nervous system is involved in different functions such as transduction of afferent sensory inputs, trophic actions, modulation of immunologic events and thermoregulation. In the present investigation, we studied the pattern of human autonomic skin innervation with special reference to its relation to blood vessels, hair follicles, sweat glands and sensory receptors. For the first time, two clinically important areas have been compared: the skin of the forearm and of the face. Using indirect immunohistochemistry, we analyzed the distribution of calretinin (CR), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), tyrosine hydroxylase (TH), histamine, serotonin, enkephalin, and, enzyme histochemically, NADPH-diaphorase (NADPH-d). In the epidermis, we found nerve fibers containing SP, NKA and CGRP. In the dermis, SP-, CR-, VIP-, CGRP- and NKA-positive nerve fibers were detected. Particularly the large nerve fibers contained CR. VIP-positive fibers occurred especially around hair follicles and sweat glands. CGRP-positive nerve fibers were located close to the epidermal basal membrane, in the wall of blood vessels, and to a lesser extent around hair follicles. Immunoreactivity for SP and NKA in the dermis was observed predominantly in the papillary layer near the epidermal basal membrane. All neuropeptides tested in this study were also detected in the nerve fibers of the subcutis. Most of them were CGRP- and VIP-positive. They occurred in association with sweat glands and large arteries. NPY-positive nerve fibers are predominant in the wall of arteries, arterioles and veins. Nerve fibers containing NKA and SP were less common and identified only in the walls of large arteries in deeper dermal layers. In double-staining experiments, the NADPH-d reaction and reactivity to tubulin revealed a partial co-localization in nerve fibers, blood vessel walls, around glands and ganglionic cells. VIP-positive fibers were more common in the face skin than in the forearm. However, in forearm we detected more NPY-, CGRP-, NKA- and SP-positive nerve fibers than in face skin. These findings are important for future studies on skin disorders, such as sensory neuropathies, inflammatory reactions or allergic responses of human skin.

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8

Abstract Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell’s osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems.

Embryonic and early fetal development of human taste buds: A transmission electron microscopical study

Taste buds are assemblies of slender epithelial cells that receive chemical stimuli from the outer (oral) environment. In contrast to the large and well documented information on the morphology of taste buds in adult humans and animals, there are only a few reports on fetal ones, and ultrastructural studies of prenatal human taste buds are lacking completely. Therefore, the present investigation has been carried out to study the taste bud primordium, its morphological changes including synaptogenesis, cell differentiation, and taste pore formation from the time of the onset of taste bud formation around the 8th week until the 15th postovulatory week.

Collagen Type I Increases Bone Remodelling around Hydroxyapatite Implants in the Rat Tibia

The early interface reaction of cancellous bone to a nanocrystalline hydroxyapatite (HA) cement containing 3 wt% collagen type I (HA/Coll) with a setting under physiological temperature and pH was observed using immunohistochemical techniques. Pure HA served as a control. Cylinders with a diameter of 2 mm were implanted into the proximal tibia of 72 adult Wistar rats. Histological sections of 6 animals were prepared after 1, 2, 4, 6, 14 and 28 days. First, osteoblast-like cells as well as a marked reaction for osteonectin, osteopontin and its ligand CD44 were observed as early as 2 days after implantation at the interface around HA/Coll implants. Further, reactivity for ED1 and cathepsin D, both markers for phagocytotic cells, appeared earlier and stronger around HA/Coll. In cell counts, a significantly higher average number of ED1- and cathepsin D-positive phagocytotic cells was observed around the HA/Coll implants on days 6 (p < 0.01), 14 and 28 (p < 0.05). The number of osteopontin-positive cells was significantly higher around HA/Coll implants at days 6 and 14 (p < 0.05). Two weeks after the implantation, first islands of newly formed woven bone were observed around the HA/Coll implant, but not around the control implant. The amount of direct bone contact after 28 days averaged 28% around pure HA and 51% around HA/Coll implants (p < 0.05). While both implants displayed a good osteoconductivity, a higher bone remodelling activity was observed around collagen-containing HA implants compared to pure HA implants. It appears that the addition of collagen to HA implants can enhance both phagocytotic and osteogenic processes. This may result in an earlier acceptance and better osseointegration of the HA/Coll implants into the surrounding tissue.

Intranasal administration of drugs.

OBJECTIVE To investigate how nasally applied substances distribute in the nose depending on the form of application. DESIGN Observer-blinded study. SETTING University hospital research unit. PARTICIPANTS Fifteen healthy volunteers aged 22 to 32 years. INTERVENTIONS Forms of application included (1) nasal drops applied with a pipette, (2) nasal spray, and (3) a system producing squirts. Blue food dye was used to visualize the intranasal distribution of the liquid. The investigation was performed using nasal endoscopy. MAIN OUTCOME MEASURE Intranasal distribution of the dye was judged by 2 independent observers blinded to the applicator system used. RESULTS The nasal drops predominantly reached the nasal floor. The nasal spray was widely distributed in the nasal mucosa; however, most of it was intercepted by the middle turbinate and did not reach the olfactory cleft effectively. Using the squirt system, the olfactory cleft was reached in most participants. CONCLUSIONS Previous failure of therapy with locally applied drugs in the case of sinonasal smell disorders may be partly due to the fact that the drugs did not reach the olfactory cleft when using traditional forms of application (ie, sprays). However, using an applicator producing squirts seems likely to present the drugs more effectively to the olfactory epithelium. Thus, it may be hypothesized that therapy could be more effective using a squirt system.

Immunohistochemical, volumetric, and functional neuroimaging studies in patients with idiopathic Parkinson's disease.

Idiopathic Parkinsons disease (PD) is closely associated with olfactory loss. Deficits in the sense of smell may precede clinical motor symptoms by years. Although there is more and more evidence from recent studies to support this view, it remains unclear which substrates would cause the olfactory deficit. Studies based on biopsies from the olfactory epithelium did not reveal specific changes in the nasal mucosa of PD patients compared to patients who were hyposmic for other reasons. Thus, PD-related olfactory impairment seems not to be directly associated with specific changes in the olfactory epithelium. With regard to volumetrics of the olfactory bulb (OB) results indicated that there is little or no difference between PD patients and healthy controls in terms of OB volume. Again, these data support the idea that olfactory loss in PD is not a consequence of damage to the olfactory epithelium but rather results from central-nervous changes. Finally, studies based on functional MRI suggested that neuronal activity in the amygdala and hippocampus is reduced in PD patients compared to controls which may specifically impact on olfactory function. In addition, neuronal activity in components of cortico-striatal loops appears to be up-regulated indicating compensatory processes involving the dopaminergic system. Thus, it seems that cerebral changes, and not changes at the level of the olfactory epithelium, are the basis of the olfactory loss observed in PD patients.

Contact Endoscopic Comparison of Morphology of Human Fungiform Papillae of Healthy Subjects and Patients with Transected Chorda Tympani Nerve

Background: The chorda tympani nerve (CTN) carries gustatory fibers from taste buds of fungiform papillae (fPap) of the anterior portion of the tongue. Accordingly, middle ear surgery with transection of the CTN may result in gustatory impairment. With use of contact endoscopy, the present study aimed to compare number and shape of fPap and subepithelial vessel formation in patients after CTN transection with that of healthy controls.

Innervation of developing human taste buds. An immunohistochemical study

Abstract Morphological changes in developing human gustatory papillae during the 6th to the 23rd postovulatory week have been studied. The general innervation pattern of taste papillae and taste bud primordia was revealed immunohistochemically using antibodies against protein gene product 9.5 (PGP9.5), neurofilament H (NFH), neurofilament L (NFL), neurone-specific enolase (NSE), and tubulin. The autonomic and somatosensory nerve supply has been investigated using antibodies against substance P (SP), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), neuropeptide Y (NPY), the neuronal form of nitric oxide synthase (n-NOS), and, enzyme histochemically, NADPH-diaphorase. Nerve fibers approach the basal membrane of the lingual epithelium around the 7th postovulatory week and invade the epithelium of papilla-like structures at the 8th week, but some also penetrate the basal membrane of the non-papillary epithelium. They are in close contact with slender epithelial cells that are considered to be the taste bud’s progenitor cells. Early human taste buds situated at the anterior part of the tongue do not necessarily require a dermal (later fungiform) papilla. The NADPH-diaphorase reaction revealed positive results in dermal nerve fibers, but the immunohistochemical reaction against n-NOS was negative. Immunohistochemical detection of neuropeptides and vasoactive substances rendered negative results for developmental stages of 7–18 postovulatory weeks. By the 18th week, only SP was detected in dermal papillae, but not in the vicinity of taste buds’ primordia. Thus, autonomic and somatosensory nerves seem not to play a key role in formation and maintenance of early human taste buds.