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In situ study of partially crystallized Bioglass and hydroxyapatite in vitro bioactivity using
atomic force microscopy
I.B. Leonor, A. Ito, K. Onuma, N. Kanzaki, Z.P. Zhong, D. Greenspan, R.L.
Reis
Department of Polymer Engineering, University of Minho, Campus
de Azurem, 4800-058 Guimaraes, Portugal
Tissue Engineering Research Center, National Institute for Advanced Industrial
Science and Technology, Central 4, 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8562,
Japan
USBiomaterials Corporation, One Progress Blvd, #23, Alachua, FL 32615
The present work investigates, in situ, the in vitro bioactivity of partially
crystallized 45S5 Bioglass® (BG) as a function of immersion
time in a simulated body fluid (SBF) using atomic force microscopy (AFM). The
results obtained for the crystallized BG were compared to those of
hydroxyapatite c- and a-faces. The calcium phosphate layer grows on the
crystallized 45S5 BG by multiple two-dimensional nucleation and fusion of these
two-dimensional islands, which is essentially the same mode as for hte
hydroxyapatite c-face. The surface of the crystallized 45S5 BG was almost
fully covered with a dense and compact calcium phosphate layer after 24 h.
The calcium phosphate formation on the crystallized BG arises from a low surface
energy of the surface layer and/or an effect of the layer to lower the
resistance when the growth units of calcium phosphate incorporate into the
growing island. These results indicate that the crystallized 45S5 BG is
suitable to be used as a filler for polymeric matrix bioactive composites, as it
maintains a high bioactivity associated with a stiffer behavior (as compared to
standard BG).
20 June 2002 in Wiley InterScience. DOI: 10.1002/jbm.10289
J Biomed Mater Res 62: 82-88,2002
Particulate Bioglass reduces the viability of bacterial
biofilms formed on its surface in an in vitro model.
Allan L, Newman H, Wilson M.
Department of Microbiology, Eastman Dental Institute, London, UK.
iallan@eastman.ucl.ac.uk
45S5 Bioglass is a bioactive implant material which, in its particulate form, is
used in the repair of periodontal defects. The surface reactions undergone by
this material in an aqueous environment may exert an antibacterial effect that
would be beneficial to periodontal surgical treatment. Biofilms of Streptococcus
sanguis, an early plaque former, and mixed species biofilms from a salivary
inoculum grown under conditions similar to those associated with periodontal
implants, were grown on particulate Bioglass in a constant depth film fermenter
(CDFF). Control biofilms were grown on inert glass particulates. At sample times
of 3, 24 and 48 hours the viability of biofilms of S. sanguis grown on Bioglass
was significantly lower than for those grown on inert glass. In the experiments
with subgingivally-modelled mixed species biofilms, the total anaerobic counts
were significantly lower on Bioglass after 24 and 48 hours, but not 96 or 168
hours, compared to inert glass. Thus, particulate Bioglass has the potential to
reduce bacterial colonisation of its surface in vivo, a feature relevant to
post-surgical periodontal wound healing.
Clin Oral Implants Res 2002 Feb;13(1):53-8
Bioactive glass stimulates in vitro
osteoblast differentiation and creates a favorable template for bone tissue
formation.
Loty C,
Sautier JM, Tan MT, Oboeuf M, Jallot E,
Boulekbache H, Greenspan D, Forest N.
Laboratoire de Biologie-Odontologie, Faculte de Chirurgie
Dentaire, Institut Biomedical des Cordeliers, Universite Paris 7, France.
In this study, we have investigated the behavior of fetal rat osteoblasts
cultured on bioactive glasses with 55 wt% silica content (55S) and on a bioinert
glass (60S) used either in the form of granules or in the form of disks. In the
presence of Bioglass granules (55 wt% silica content), phase contrast microscopy
permitted step-by-step visualization of the formation of bone nodules in contact
with the particles. Ultrastructural observations of undecalcified sections
revealed the presence of an electron-dense layer composed of needle-shaped
crystals at the periphery of the material that seemed to act as a nucleating
surface for biological crystals. Furthermore, energy dispersive X-ray (EDX)
analysis and electron diffraction patterns showed that this interface contains
calcium (Ca) and phosphorus (P) and was highly crystalline. When rat bone cells
were cultured on 55S disks, scanning electron microscopic (SEM) observations
revealed that cells attached, spread to all substrata, and formed multilayered
nodular structures by day 10 in culture. Furthermore, cytoenzymatic localization
of alkaline phosphatase (ALP) and immunolabeling with bone sialoprotein antibody
revealed a positive staining for the bone nodules formed in cultures on 55S. In
addition, the specific activity of ALP determined biochemically was
significantly higher in 55S cultures than in the controls. SEM observations of
the material surfaces after scraping off the cell layers showed that mineralized
bone nodules remained attached on 55S surfaces but not on 60S. X-ray
microanalysis indicated the presence of Ca and P in this bone tissue. The
55S/bone interfaces also were analyzed on transverse sections. The interfacial
analysis showed a firm bone bonding to the 55S surface through an intervening
apatite layer, confirmed by the X-ray mappings. All these results indicate the
importance of the surface composition in supporting differentiation of
osteogenic cells and the subsequent apposition of bone matrix allowing a strong
bond of the bioactive materials to bone.
J Bone Miner Res 2001 Feb;16(2):231-9
Antibacterial activity of particulate bioglass against supra- and
subgingival
bacteria.
Allan I, Newman H, Wilson M.
Department of Microbiology, Eastman Dental Institute, University
College London, UK. iallan@eastman.ucl.ac.uk
Particulate Bioglass is a bioactive material used in the repair of periodontal
defects. This material undergoes a series of surface reactions in an aqueous
environment which lead to osseointegration. The aim of this study was to
determine whether these reactions exerted an antibacterial effect on a range of
oral bacteria. Streptococcus sanguis, Streptococcus mutans and Actinomyces
viscosus were suspended in nutrient broth (NB), artificial saliva (AS) or
Dulbecco's modified eagle medium plus 10% foetal calf serum (DMEM + 10%FCS),
with or without particulate Bioglass. All bacteria showed reduced viability
following exposure to Bioglass in all the media after 1 h. This antibacterial
effect increased after 3 h. Porphyromonas gingivalis, Fusobacterium nucleatum,
Prevotella intermedia and Actinobacillus actinomycetemcomitans were suspended in
either BM broth or 40% horse serum (HS) in RPMI. A considerable reduction in
viability was observed with all bacteria tested, in both media, compared to
inert glass controls. In further experiments it was found that the viability of
S. sanguis was significantly reduced following exposure to NB pre-incubated with
Bioglass. Additionally, it was found that neutralisation of this highly alkaline
solution eliminated the antibacterial effect. Moreover, a solution of NB and
NaOH (of equivalent pH) exerted an antibacterial effect of similar magnitude to
that of the solution pre-incubated with Bioglass. Thus, particulate Bioglass
exerts an antibacterial effect on certain oral bacteria, possibly by virtue of
the alkaline nature of its surface reactions. This may reduce bacterial
colonisation of its surface in vivo.
Biomaterials 2001 Jun;22(12):1683-7
The Evaluation of
Surface Structure of
Bioactive Glasses In Vitro
Greenspan DC, Zhong JP, LaTorre GP
USBiomaterials Corporation, One Progress Blvd, #23,
Alachua, FL 32615, Advanced Materials Research Center, University of Florida,
One Progress Blvd, #14, Alachua, FL 32615
Evaluation of the surface structure of four compositions of
bioactive glasses was conducted in this work. The formation of an hydroxyl
carbonate apatite (HCA) surface layer on the bioactive glasses reacted in a tris
buffer solution appeared to be related to the development of a high surface
area. It was found that a surface area of at least 40 to 60 m2/g
was needed to induce the precipitation and crystallization of HCA. For the
bioactive glass which can bond to either bone or soft tissue, the surface area
ultimately attained was greater than 100 m2/g. These
in-vitro results could provide useful information for further investigation
of bonding of biomaterials with living tissue.
Ionic products of bioactive
glass dissolution increase proliferation of human
osteoblasts and induce insulin-like growth factor II mRNA expression and protein
synthesis.
Xynos ID, Edgar AJ, Buttery LD, Hench LL, Polak JM.
Department of Histochemistry, Imperial College School of Science, Technology and
Medicine, London, W12 ONN, United Kingdom.
Bioglass 45S5 is an osteoproductive material, which resorbs by releasing its
constitutive ions into solution. Treatment with the ionic products of Bioglass
45S5 dissolution in DMEM for 4 days increased human osteoblast proliferation to
155% of control. Two days after treatment, differential gene expression was
analyzed by cDNA microarrays. Expression of a potent osteoblast mitogenic growth
factor, insulin-like growth factor II (IGF-II), was increased to 290%.
Additionally, there was a 168% increase in the concentration of unbound IGF-II
protein in the conditioned media of treated osteoblasts. Expression levels of
IGFBP-3, an IGF-II carrier protein, metalloproteinase-2 and cathepsin-D were
also increased to 200, 340, and 310% of control levels, respectively.
Metalloproteinase-2 and cathepsin-D are proteases that cleave IGF-II from its
carrier proteins, resulting in the release of the unbound biologically active
IGF-II. We suggest that the stimulatory effect of the ionic products of Bioglass
45S5 dissolution on osteoblast proliferation may be mediated by IGF-II.
Copyright 2000 Academic Press.
Biochem Biophys Res Commun 2000 Sep 24;276(2):461-5
Bioglass 45S5 stimulates
osteoblast turnover and enhances
bone formation In vitro: implications and applications for bone tissue
engineering.
Xynos ID, Hukkanen MV, Batten JJ, Buttery LD, Hench LL, Polak
JM.
Department of Histochemistry, Commonwealth Building, Imperial
College School of Medicine, Hammersmith Campus, The Hammersmith Hospital, Ducane
Rd, London W12 ONN, UK.
We investigated the concept of using bioactive substrates as templates for in
vitro synthesis of bone tissue for transplantation by assessing the osteogenic
potential of a melt-derived bioactive glass ceramic (Bioglass 45S5) in vitro.
Bioactive glass ceramic and bioinert (plastic) substrates were seeded with human
primary osteoblasts and evaluated after 2, 6, and 12 days. Flow cytometric
analysis of the cell cycle suggested that the bioactive glass-ceramic substrate
induced osteoblast proliferation, as indicated by increased cell populations in
both S (DNA synthesis) and G2/M (mitosis) phases of the cell cycle. Biochemical
analysis of the osteoblast differentiation markers alkaline phosphatase (ALP)
and osteocalcin indicated that the bioactive glass-ceramic substrate augmented
osteoblast commitment and selection of a mature osteoblastic phenotype. Scanning
electron microscopic observations of discrete bone nodules over the surface of
the bioactive material, from day 6 onward, further supported this notion. A
combination of fluorescence, confocal, transmission electron microscopy, and
X-ray microprobe (SEM-EDAX) examinations revealed that the nodules were made of
cell aggregates which produced mineralized collagenous matrix. Control
substrates did not exhibit mineralized nodule formation at any point studied up
to 12 days. In conclusion, this study shows that Bioglass 45S5 has the ability
to stimulate the growth and osteogenic differentiation of human primary
osteoblasts. These findings have potential applications for tissue engineering
where this bioactive glass substrate could be used as a template for the
formation of bioengineered bone tissue.
Calcif Tissue Int 2000 Oct;67(4):321-9
Human osteoblast-like cells
(MG63) proliferate on a
bioactive glass surface.
Price N, Bendall SP, Frondoza C, Jinnah RH, Hungerford DS.
Department of Orthopaedic Surgery, Johns Hopkins Medical School,
Good Samaritan Hospital, Baltimore, Maryland 21239, USA.
Bioglass, a resorbable glass, previously has been evaluated as a bone graft
substitute using cells of animal origin. Limited information is available on its
effect on human cells. The objective of this study was to test the hypothesis
that Bioglass supports viability and proliferation of human bone cells. As a
prototype of human bone cells, the osteoblast cell line MG63 was used and
propagated on Bioglass disks. MG63 cells also were seeded onto disks made of
titanium (Ti-6Al-4V) and of cobalt chrome (Co-Cr-Mo) alloys. The number of
viable cells recovered was similar for Bioglass, titanium, and polystyrene
control surfaces. Significantly fewer cells were recovered from CoCr (P < 0.05)
compared to Bioglass, Ti-6 Al-4v, and polystyrene surfaces. The proportion of
cells undergoing DNA synthesis, estimated by thymidine uptake, was significantly
greater on Bioglass and titanium surfaces (P < 0.05) than on the CoCr surface.
There were detectable differences in cell morphology on these biomaterials.
Functional capacity was tested by assay of osteocalcin production and no
differences were detectable among the different biomaterials. This study
supports the hypothesis that 45S5 Bioglass provides a favorable environment for
human osteoblast proliferation and function. Bioglass may have clinical
potential as a bone graft substitute, a bioactive grout, or an implant coating
for promoting bony ingrowth in uncemented prostheses.
J Biomed Mater Res 1997 Dec 5;37(3):394-400
Histological and
biochemical evaluation of osteoblasts
cultured on bioactive glass, hydroxyapatite, titanium alloy, and stainless
steel.
Vrouwenvelder WC, Groot CG, de Groot K.
Department of Biomaterials, University of Leiden, The
Netherlands.
We investigated the behavior of fetal rat osteoblasts cultured on four bone
replacing materials: bioactive glass, hydroxylapatite, a titanium alloy, and
stainless steel. The cultures were histologically examined for individual cell
morphology and osteoblast expression after several periods of time using
scanning electron, fluorescence, and normal light microscopy. Other cultures
were used for biochemical determinations of alkaline phosphatase activity (APA)
and DNA content. Osteoblasts cultured on bioactive glass showed a better
osteoblast-like morphology and a higher proliferation rate, leading to confluent
cultures with higher cell density and a generally better expression of the
osteoblast phenotype in comparison with the other substrates. The confluent
bioactive glass cultures also showed significantly higher DNA content and APA as
well as the calculated APA/DNA ratio. Based on the evaluation of histological
and biochemical parameters we conclude that osteoblasts cultured on bioactive
glass show a generally better osteoblast character than on the other materials.
J Biomed Mater Res 1993 Apr;27(4):465-75
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