Titanium implants with two different surfaces: Histomorphologic and histomorphometric evaluation in rabbit tibia.

This study aimed to compare two different implant surface treatments of the implant system Bi-Implant (Plan 1 Health): one surface sandblasted with hydroxyapatite (HA) (Osseogrip(R)) and one machined surface. Histomorphologic and histomorphometric evaluations of the bone healing at the interface between a titanium implant and bone were performed using a light microscopic technique. Twenty-four commercially pure titanium implants with a smooth surface and 24 implants with a sandblasted surface were inserted in the tibias of 12 rabbits. The 12 rabbits were divided into three groups, each consisting of four animals, were sacrificed at 4 weeks (I group), 8 weeks (II group) and 12 weeks (III group) after the insertion of the bio-material. The results emphasized that in the sections examined with the light microscope, the bone was in intimate contact with the implant surface and the bone surrounding the implants was mostly lamellar. After 4 weeks, mature bone tightly surround-ing some areas of the implant perimeter was observed. The implant with the Osseogrip(R) surface showed an average percentage of bone-implant contact (%BIC) equal to 33%, while the one with the machined surface showed a %BIC equal to 17%. After 8 weeks, a progressive increase in mineralized bone surrounding the implant surface was detected, making the results of the machined surface superposable to the Osseogrip(R) surface results (48 and 44%). After 12 weeks, the implants with the machined surface exhibited close contact with the bone tissue corresponding to 62% of their perimeter, while for the implants with the Os-seogrip(R) surface the surface contact was 67% of the implant surface. The morphometric evaluation of %BIC at the three time points evidenced an increase in bone-titanium contact over time on both machined and Osseogrip(R) surfaces. Moreover, implants with rough surfaces demonstrated better behavior than the implants with the machined surface when taking into account the earlier osteointegration (4 weeks) of the peri-implantar tissues.

The adhesion of Flow 2002 fibroblasts to titanium implant materials is influenced by different surface topographies and is related to the immunocytochemical expression of fibronectin.

Osteointegrated titanium dental implants are widely used biomaterials that have to integrate within the alveolar bone and interact with periodontal soft tissues. In this study, we investigated the immunocytochemical expression of the extra-cellular matrix (ECM) protein fibronectin (FN) and type I collagen (Coll I) in Flow 2002 fibroblast cultures spread on grade III-titanium samples with five different surface topographies and we correlated the immunocytochemical data to the adhesion capability of these cells to the above-mentioned substrates. Five different surfaces of grade III-titanium implants were at first characterized both by scanning electron microscopy (SEM) and by laser profilometry for surface roughness evaluation. After being spread on the biomaterial surfaces, the fibroblasts were left to proliferate for 72 hr and subsequently the cells underwent immunocytochemical procedures for detecting both FN and Coll I. The fibroblasts appeared more adherent to smoother titanium surfaces than to rougher ones; however, the highest cell density was detected on the roughest surface, even if it was unrelated to the highest FN expression. In the other biomaterial surfaces examined, as well as in controls, immunocytochemical FN expression correlated effectively to cell density on the examined substratum, whereas no determinant information was available regarding Coll I. It is reasonable to assume that surface roughness could be a relevant parameter influencing fibroblast adhesion to substrata; however, the evaluation of the cell density only is insufficient, and should be supported by the immunocytochemical FN expression, which could be confirmed as a useful tool in determining implant material biocompatibility. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 169-76).

Porous titanium obtained by a new powder metallurgy technique: Preliminary results of human osteoblast adhesion on surface polished substrates.

This study concerns a novel powder metallurgy method for producing porous titanium (pTi) exhibiting high mechanical properties. The preparation procedure consisted of the following stages: first, the preparation of Ti and titanium hydride (TiH2) powder mixtures and their consolidation with a cold isostatic press, followed by a sintering of the green bodies performed with hot isostatic press (HIP) equipment. Thermal decomposition in controlled environment of the TiH2 phase results in the foam structure. The resulting porosity percolates with a volume fraction of approximately 20%. The final material exhibits interesting mechanical properties, comparable to those of full density titanium (between grade 2 and grade 3), with the advantage of a minor density. The samples produced were tested to verify their biological response by studying the effectiveness of osteoblast adhesion and growth. In this preliminary study, osteoblastic cell morphology was investigated and compared to that observed on fully dense commercially pure titanium (Ti-cp) (ASTM, grade 3). The preliminary results were promising regarding cellular adhesion and spreading. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 172-7).

Morphological features and measurements in a human fetus with karyotype 69, XXX: comparison with fetuses of the same CRL, without chromosomal anomalies.

A triploid fetus (karyotype 69, XXX) with crown-rump length (CRL) 94 mm, presenting micro- and retrognathia, low-set ears and crooked feet, was cleared and double-stained with alizarin red S and alcian blue for detecting the ossification patterns in the vertebral column, ribs, ischium, limbs, and face. Longitudinal measurements of some long bones in the upper (humerus, ulna, radius) and lower (femur, tibia, fibula) limb were taken. The values of both the total length (TL) and the ossified part (OL) of each long bone, as well as the OL/TL per cent ratio were considered. Reference points were located on the mandible, i.e. condylar process (Pcl), coronoid process (Pco), gnathion (GN), gonion (GO), superior symphyseal point (SSP) for measuring linear dimensions. Since the aim of this work was to assess the influence of triploidy 69, XXX the skeletal development and growth patterns, all values obtained in the examined specimen were related with those relative to a group of fetuses, without any detectable malformation and chromosomal anomalies, with a CRL mean value of 93 mm. Results evidenced that the triploid fetus presented growth restriction and that the vertebral centra ossification and the mandibular development were much delayed with the normal ossification patterns.

Immunocytochemical expression of protein kinase C isoenzymes alpha, delta, epsilon and zeta in differentiating chick chondrocytes in vitro.

In our previous work we have investigated the expression of the serine-threonine kinase protein kinase C (PKC) in the vertebral column of mouse foetuses. In the present work we would verify the expression of four PKC-isoenzymes (alpha, delta, epsilon, zeta) in two distinct phases of the chondrogenesis and the endochondral osteogenesis in vitro. We performed primary cultures of chondrocytes collected from tibiae of 6-day old chick embryos. This cells were cultured for 20 days and than collected on coverslips (stage 1 culture). Other cells of the stage 1 were undergone further differentiation towards the phenotype of osteoblast-like cells (stage 2 culture), in accord to the protocol of Descalzi Cancedda et al. (1992). In stage 1 culture, PKC-epsilon was the most expressed isoform, whereas PKC-alpha exhibited the least intense positivity. In stage 2 culture, PKC-alpha was the most expressed isoform, whereas a marked decrease of PKC-epsilon expression was detected compared to stage 1. No relevant differences were evidenced as regards,the expression of PKC-zeta between the two considered cell culture stages. On these bases, it could be reasonable that these PKC-isoenzymes may be involved at different levels in chondrocytes differentiation as well as in the endochondral ossification process.

Biocompatibility evaluation of dental metal alloys in vitro: expression of extracellular matrix molecules and its relationship to cell proliferation rates.

The biocompatibility in vitro of dental biomaterials has been widely studied, with consideration of cell viability and cell proliferation rates. In the present study we evaluated the biocompatibility in vitro of three single-phase dental metal alloys, all provided by the same manufacturer. To this aim, we considered the percentage of proliferating cells revealed by 5-bromodeoxyuridine incorporation in human fibroblast cultures in the presence of these biomaterials, performing a short time test (72 h). These data were correlated with immunocytochemical expression of four molecules of the extracellular matrix, i.e., fibronectin, type I collagen, beta(1)-integrin subunit, and chondroitin sulfate, because the capability of cells to adhere to substrata is widely related to cell proliferation rates. Alloys presenting higher amounts of noble elements were more biocompatible even when they contained significant amount of both Ag and Cu. As regards the expression of the extracellular matrix molecules, the organization level of fibronectin in fibrils was correlated with higher cell proliferation rates, whereas no difference was detected for the expression of the other antigens. On these bases, we assume that expression of fibronectin could be a useful parameter in evaluation of biocompatibility in addition to cell proliferation capability.

Cell proliferation rates and fibronectin arrangement as parameters for biocompatibility evaluation of dental metal alloys in vitro.

A short-term (72-96 hours) biocompatibility evaluation in vitro of four single phase dental metal alloys was conducted by determining cell proliferation rates correlated to the organization of the extracellular matrix protein fibronectin in human fibroblast cultures. Immunocytochemical methods were performed to detect both cell proliferation rates by 5-bromodeoxyuridine (BrdU) incorporation, and fibronectin arrangement, i.e., diffuse in the extracellular matrix, organized in fibrils or in focal adhesions. We showed that cell proliferation rates were related to fibronectin expression. In particular, a higher percentage of cells in the S-phase were related to a predominance of fibronectin organized both in fibrils and in focal adhesions. The alloy with the highest Au content seemed the most biocompatible among those tested, since it behaved in a very similar manner to the controls. On the contrary, fibroblasts exposed to the alloy with the highest percentage of Ag had the most different behavior as compared to the controls. We can assume that a correlation exists between fibronectin organization and the percentage of BrdU-positive cells and that these parameters are varying with the different metal composition of the alloys. The observation of fibronectin arrangement together with cell proliferation rates could be considered a useful tool to determine the biocompatibility of these biomaterials.

In vitro evaluation of the biocompatibility of dental alloys: fibronectin expression patterns and relationships to cellular proliferation rates.

OBJECTIVE: This short-term (72- to 96-hour) in vitro study on fibroblasts evaluated the biocompatibility of 3 single-phase dental alloys by determining cellular proliferation rates and the expression of a glycoprotein, fibronectin, which is involved in cellular adhesion processes. METHOD AND MATERIALS: Flow 2002 fibroblasts were cultured together with 3 single-phase dental alloys of different composition. Proliferation rates were determined by 5-bromodeoxyuridine incorporation. Fibronectin expression was determined by indirect immunofluorescence. RESULTS: At 72 hours, cells cultured with the alloy containing the lowest amount of noble elements (gold, platinum, and palladium) and the highest amount of silver exhibited significantly less proliferation than did controls. At 96 hours, only cultures with the alloy containing the greatest amount of noble elements behaved in a way similar to controls. Fibronectin organization in fibrils and in focal adhesions was correlated to higher cellular proliferation rates. CONCLUSION: Fibronectin organization could be a useful tool to determine the biocompatibility of dental alloys. Among the noble elements, palladium by itself exhibits very good biocompatibility. These indications could be useful for practitioners in the choice of the best alloy for specific clinical applications.

Biocompatibility in vitro of titanium dental implants. Immunocytochemical expression of fibronectin and extracellular matrix receptors.

BACKGROUND: Cell-substratum interactions play a peculiar role in cell proliferation, differentiation and migration. They are regulated by various glycoproteins, among which fibronectin, and by receptors connecting cells to the extracellular matrix, i.e. integrins. Therefore, the aim of this study was to correlate the proliferation rates of the human fibroblast line WI-38 cultured in presence of titanium dental implants to cell adhesion capability to substrata. METHODS: WI-38 fibroblasts were cultured in presence of four dental implants in titanium (one hydroxyapatite coated) for 48, 72 and 96 hours. Cell proliferation was evaluated by detecting 5-bromodeoxyuridine incorporation. Fibronectin organization and alpha5beta1 integrin expression were evidenced by indirect immunofluorescence. RESULTS: A correlation between fibronectin organization and cell proliferation rates was demonstrated: cultures showing fibronectin mainly organized in fibrils presented the highest cell proliferation degrees. After 96 hours, the observed decrease of the number of proliferating cells corresponded to a different fibronectin organization. In presence of the hydroxyapatite coated implant, colocalization of fibronectin and alpha5beta1 integrin was represented in focal contacts in cultures exhibiting the highest proliferation rate, while cells with the lowest proliferation one expressed alpha5beta1 integrin in point contacts. CONCLUSIONS: Evidences obtained in this work showed that both the organization of fibronectin and the expression of alpha5beta1 integrin are strictly correlated to cell proliferation rates. Therefore, these parameters could be useful for evaluating the biocompatibility of dental materials in vitro.

The influence of dental metal alloys on cell proliferation and fibronectin arrangement in human fibroblast cultures.

The biocompatibility of six single-phase dental metal alloys was studied by determining cell proliferation rates correlated to the arrangement of fibronectin (FN) in fibroblast cultures. Immunocytochemical methods were used to detect cell proliferation by 5-bromodeoxyuridine (BrdU) incorporation, and FN organization [i.e. diffuse in the extracellular matrix and organized in fibrils or in focal adhesions (FA)] in human fibroblast cultures. Cell proliferation rates were related to FN arrangement and in particular a higher percentage of cells in the S-phase was related to a predominance of FA. The greatest difference in behaviour compared to that of the controls was detected after 120 and 168 hr: at these times, as well as at previous ones, the alloy with the highest Au content seemed the most biocompatible among those tested, as it behaved in a very similar way to the controls. In contrast, fibroblasts exposed to the other five alloys showed different behaviours from the controls. It is assumed that a correlation exists between FN organization and the percentage of BrdU-positive cells, and that these features vary in the presence of different alloys. The observation of FN arrangement together with cell proliferation rates could be another useful tool in determining the biocompatibility of dental metal alloys.

On the assessment of the growth patterns in human fetal limbs: longitudinal measurements and allometric analysis.

The total length (TL) and length of the ossified part (OL) of some long bones of the upper (humerus, ulna, radius) and lower limb (femur, tibia, fibula) were evaluated in 58 aborted human fetuses (crown-rump length, CRL, between 38 and 116 mm, developmental age from 8 to 14 weeks). The specimens, without any detectable malformation, were cleared and double-stained with alcian blue and alizarin red S to obtain a differential detection of the ossified part within the comprehensive outline between the cartilaginous epiphyses. The correlation between the values of TL and OL and those of CRL emphasized that the systematic OL measurement in limb long bones correlated better than TL with development age, since OL increased faster than TL. TL and OL also correlated with the CRL by bivariate allometry (ln y = ln a + b ln x) and the data obtained showed that they grew with positive allometry. The comparison between the cumulative values of the bones examined in each limb showed that both TL and OL grew relatively faster in the lower limb than the upper; the greatest growth rate was found for OL in the lower limb. These results many provide a tool for a comprehensive assessment of long bone growth patterns and may be useful in determining fetal growth even in incomplete specimens, in which one or some long bones can still be measured.

The presence of implant materials influences fibronectin arrangement and cell growth in fibroblast cultures.

The nature of the bone-implant interface has been much focused in investigating dental implant materials, whereas the relationship between implant and fibroblasts has received much less attention. To evaluate the biocompatibility degree of an implant material, both cell adhesion and cell growth must be tested in the presence of the implant. Four dental implant (A,B,C,D) made in titanium alloy and one of them (C) hydroxyapatite (HA)-coated in fibroblast cultures (48 and 72 h) were tested, by performing immunocytochemical techniques and then by observing fibronectin (FN) arrangement for cell adhesion and counting 5-bromodeoxyuridine (5-BrdU) to evaluate cell proliferation. Different FN arrangements were observed-i.e. organized in fibrils, or in focal adhesion plaques, as well as dispersed in the intercellular space-which varied for the different implants employed at the various culture stages. Equally, the per cent ratio of 5-BrdU positive cells was different, with a more significant increase (p < 0.001) between 48 and 72 h for implant C and the controls. It was observed that the higher percentages of 5-BrdU positive cells were in cultures where FN was organized mainly in focal adhesions, as well as 5-BrdU positive cells increased after 72 h in cultures, which after 48 h presented much FN dispersed in the intercellular space. It may be assumed that a correlation exists between FN arrangement and the percentage of 5-BrdU positive cells and that these two parameters vary in the presence of the different implants. Moreover, the HA-coated implant seems to be the most biocompatible in fibroblast cultures.

Protein kinase C (PKC) isoenzymes exhibit specific expression in the vertebral column of human fetuses.

Recent studies have shown that protein kinase C (PKC) plays a pivotal role in many cellular functions, i.e. cell proliferation and differentiation, exocytosis, ion-exchange regulation, hormone and neurotransmitter release, programmed cell death. Up to now the tissue and organ distribution of PKC isoenzymes have been studied in various mammalian adults and it has been suggested that each of them could play a specific role in the regulation of various cellular functions. However, few works have been performed on the expression of the enzyme in actively proliferating and differentiating tissues, i.e. during embryonal and fetal developmental stages. In the present study we have performed an immunohistochemical analysis by using polyclonal antibodies in order to verify the distribution of nine PKC isoenzymes in the vertebral column of human fetuses in a key period (8th developmental week), when the most relevant chondrogenetic and osteogenetic processes occur. The detection of the various PKC isoenzymes and their different distribution in the vertebral bodies and in the intervertebral spaces lead to speculate that the appearance, localization and activation of PKC isoforms in chondrocytes might depend on the stage of chondrogenesis and suggest that cartilage and developing bone represent an appropriate cell system to understand the mechanism of signal transduction, which involves the enzyme.

Mandibular growth rates in human fetal development.

A morphometric analysis of changing proportions in the developing mandible was undertaken in 18 human embryos and fetuses of both sexes (developmental age from 8 to 14 weeks, crown-rump length, CRL, from 34 to 110 mm), previously cleared and stained with a specific method for bone (alizarin red S). Reference points were located on the mandible, i.e. condylar process (Pcl), coronoid process (Pco), gnathion (GN), gonion (GO), superior symphyseal point (SSP), for measuring linear dimensions, i.e. Pcl-GN, Pcl-Pco, Pco-GN, GO-GN, SSP-GN. The gonial (Pcl-GO-GN) and the (Pcl-GN-Pcl) angles were also measured. All linear dimensions were correlated with the CRL by bivariate allometry (1n y = 1n a+b 1n x): they all grew with positive allometry, except GO-GN with isometry. The mandibular ramus grew relatively faster than the body, both in length and height, and the greatest growth rate was found for ramus height. The relation between mandibular shape and the craniofacial structures was investigated using scale drawings obtained from photographs of fetal skulls in lateral view. In the youngest fetuses the mandible was prognathic, then became retrognathic. During the period investigated the zygomatic process and squama of the temporal bone were in a lower and more inclined position in relation to the transverse plane passing through the zygomatic arch than in the newborn and adult. This study identifies parameters fitting changing trends in height, length and shape of the human mandible during the prenatal period (8-14 weeks); moreover, it emphasizes that the mandibular growth patterns differ significantly from those of successive development periods.

On the presence of a secondary cartilage in the mental symphyseal region of human embryos and fetuses.

The presence of a secondary cartilage in the mental symphyseal region was examined in this study. A double-staining method with alcian blue and alizarin red S was performed on both whole human embryos and fetuses (developmental age between 8 and 17 weeks, crown-rump length, CRL, between 37 and 124 mm) and their disjointed mandibles. Histological and histochemical techniques were applied to transverse serial sections of whole disjointed fetal heads. The ossification process observed in the mental symphysis is quite different from that of the mandibular body, whose membranous ossification is induced by the contiguous Meckel's cartilage. No evidence of any fusion of Meckel's cartilage with the symphyseal cartilage, that lies within the symphyseal space, was detected. On the basis of these findings, we suggested that the mental secondary cartilage is able to change into bone according to an endochondral ossification process. Moreover, the role of mechanical causes in the development of the mental symphysis was hypothesized.

The growth of long bones in human embryological and fetal upper limbs and its relationship to other developmental patterns.

Measurements were made of the long bones of the upper limbs (humerus, ulna, radius) of 58 aborted embryos and fetuses, developmental age from 8 to 14 weeks, crown-rump length (CRL) between 38 and 116 mm. The specimens were cleared and double-stained, using alcian blue and alizarin red S for a differential detection of cartilage and bone. The values of both the total length (TL) and the ossified part (OL) of each long bone were related to the fetal developmental age previously estimated by freshly measured CRL. The relationship to another developmental pattern, i.e. the number of ossified centres in the vertebral column, suggested that the OL values could be much more significant than TL for the assessment of fetal growth.

Lamellar and "club-shaped" corpuscular nerve endings in human gingival mucosa. A light and electron microscopic study.

A study on the presence of corpuscular nerve endings in human gingival mucosa was performed using both light and transmission electron microscopic (TEM) techniques. Both round and oval lamellar corpuscles were detected by light microscopy. They were located either subepithelially, close to the basement membrane, or within the papillae, deeply invaginated into the overlying epithelium. TEM techniques showed convoluted structures with unmyelinated fibre arborizations leading to an afferent fibre supported by the so called lamellar cells. The presence of blood vessels, collagenous fibrils, desmosome-like junctions, cytoplasmic organelles, as well as the similarity with some previously described mechanoreceptors, suggested the role of such corpuscular nerve endings in transmitting a nervous impulse induced by mechanical stimulation. Other simpler structures were also observed and named "club-shaped" corpuscles: they could support the more complex ones in responding to the strengths and the movements directly influencing the gingival mucosa.

Developmental pathways of vertebral centra and neural arches in human embryos and fetuses.

The ossification pathways of both vertebral centra (i.e., vertebral bodies) and neural arches were studied in human embryos and fetuses (CR-length between 38 and 116 mm). A clearing and double-staining method for whole embryo or fetus, using alcian blue and alizarin red S, allowed an easy and precise detection of the morphology of the whole vertebral column and every single vertebra. Both cartilaginous and bony components were clearly visible. Different temporal and topographical patterns of ossification were shown for the centra and arches; the latter were respectively proximal-distal (i.e., bidirectional from a defined starting tract in T10-L1) and cranial-caudal (i.e., monodirectional). The patterns could be related to the morphogenetic processes of other structures (i.e., muscles and nerves). Moreover, the numerical survey of ossification centers provided a possible parameter for the determination of the fetal developmental age. This could be useful in the study of pathological conditions.