Surface reactivity is one of the common characteristics of bone bioactive ceramics. It contributes to their bone bonding ability and their enhancing effect on bone tissue formation. During implantation, reactions occur at the material-tissue interface that lead to time-dependent changes in the surface characteristics of the implant material and the tissues at the interface. This review describes some of the current concepts regarding the surface reactivity of bone bioactive materials and its effect on attachment, proliferation, differentiation and mineralization of bone cells.
Bioactive glass (BG) is an effective synthetic bone graft material.
BG granules of narrow size range (300-355 mm) have the ability to form
new bone tissue inside excavations produced by in vivo resorption
Previously,
we demonstrated that BG stimulates the differentiation of cultured
osteoblast
precursors if the glass surface was biomimetically modified by
formation
of bone-like apatite and adsorption of serum proteins, We now report
that
modified BG can also increase the rate at which multipotential rat bone
marrow stromal cells (rMSC) will undergo osteogenesis. BG promoted rMSC
osteogenesis both when cells were plated in contact with BG and when
cells
were not directly in contact with the BG.
Alkaline phosphatase activity, a marker of bone cell
differentiation,
was used as an indicator for osteogenesis. Alkaline phosphatase
activity
of rMSCs exposed to osteoinducers such as ascorbate, dexamethasone and
BMP-2, was enhanced in the presence of BG. The stimulatory effect of BG
was more pronounced in rMSC cultures with low basal alkaline
phosphatase
activity than in those with higher activity. The enhanced
differentiation
of rMSCs was associated with both a change in rMSC morphology and
altered
chemical composition of the cell culture media. rMSCs cultured on BG in
the presence of BMP or dexamethasone exhibited a more rounded
osteoblast-like
appearance as compared with cells grown on tissue culture plastic. In
the
presence of BG, elevated levels of calcium and silicon in the culture
medium
were observed throughout the 7 day culture period, suggesting a
continuous
dissolution of surface modified BG and resulting release of BG
dissolution
products. The data suggest that both surface- and solution-mediated
events
play a role in the osteogenic effect of BG.
The contribution of non-specific interactions between cells and model functional surfaces was measured using a spinning disc apparatus. These model functional surfaces were created using self-assembled monolayers (SAM) of alkylsilanes terminated with epoxide, carboxyl (COOH), amine (NH(2)), and methyl (CH(3)) groups. These SAMs were characterized using ellipsometry, atomic force microscopy, contact angle goniometry, and X-ray photoelectron spectroscopy to confirm the presence of well-formed monolayers of expected physicochemical characteristics. All substrates also demonstrated excellent stability under prolonged exposure (up to 18h) to aqueous conditions. The adhesion strength of K100 erythroleukemia cells to the functional substrates followed the trend: CH(3) < COOH ~ epoxide << NH(2). The NH(2) SAM surface exhibited nearly an order of magnitude greater adhesion strength than the other SAMs and this non-specific effect exceeded the adhesion measured when RGD tri-peptides were also immobilized on the surface. These findings illustrate the importance of substrate selection in quantitative studies of peptide-mediated cellular adhesion.
Bioactive glass granules were implanted in the tibiae of rabbits in order to determine the pathway of the silicon released from bioactive glass in vivo. We traced and quantified the silicon released by obtaining 24-h urine samples and blood samples for up to 7 months after implantation. Bone tissue as well as the following organs were resected for chemical and histopathological analyses: kidney, liver, lung, lymph nodes, and spleen. The urinary silicon of the implanted group was significantly higher than in the control group. From the data, the calculated average excretion rate was approximately 1.8 mg/day, and as such, the amount of implanted silicon was excreted within statistical bounds in 24 weeks. At this point, only elevated concentrations of silicon were found at the implant site and not in the other organs. The concentrations of silicon measured in the urine were well below saturation. Since no significant increase in silicon was found in any of the organs including the kidney, the increased silicon excretion rate was within the physiological capacity of rabbits. Therefore, it can be concluded that the resorbed silica gel is harmlessly excreted in soluble form through the urine.
Previous studies indicate that surface modification of porous bioactive glass (BG) promotes osteoblast function. We hypothesize that bone formation on treated BG is due to the selective adsorption of serum attachment proteins. To test this hypothesis, we examined the profile of proteins adsorbed onto treated BG and compared these proteins with those adsorbed to untreated BG and porous hydroxyapatite (HA). Porous BG was treated with Tris buffered electrolyte solution so as to generate a calcium phosphate-rich (Ca-P) surface layer, and then immersed in tissue culture medium containing 10% serum. Proteins adsorbed on the ceramic surfaces were analyzed by SDS-PAGE and Western blot analysis. Porous HA bound a higher amount of total protein than the other substrates. However, surface modified porous BG adsorbed more fibronectin than HA. The effect of serum protein adsorption on osteoblast adhesion to BG and HA was also evaluated. Cell adhesion to surface modified and serum-treated porous BG was significantly greater than to either surface modified or serum treated porous BG. Furthermore, cell adhesion to porous BG treated to form the dual layer of Ca-P and serum protein was significantly higher than to HA with adsorbed serum protein. Results of the study strongly suggest that adsorption of serum fibronectin onto the surface of modified porous BG, which is coated with Ca-P, may be responsible for enhanced osteoblast adhesion.
SANTOS E, RADIN S, SHENKER B, SHAPIRO I, DUCHEYNE
P
SI-CA-P XEROGELS AND BONE MORPHOGENETIC PROTEIN ACT SYNERGISTICALLY
ON RAT STROMAL MARROW CELL DIFFERENTIATION IN VITRO.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 41(1):87-94, 1998 JUL.
This study describes a novel bioactive xerogel glass as a carrier for bone morphogenetic protein (BMP) and the value of this carrier in terms of stimulating osteogenic activity of rat stromal marrow cells in vitro. These cells were seeded onto the surface of xerogel glass discs with BMP either incorporated in the glass, adsorbed to the surface of the glass or added to the culture media and compared to cells on glass with no added BMP, or cells on tissue culture plastic (TCP) with and without BMP. Cells were cultured for six and ten days and examined for total DNA, alkaline phosphatase activity, and osteocalcin and total protein production. Stromal cell differentiation, as measured by alkaline phosphatase activity and osteocalcin synthesis was most increased when the BMP was incorporated or adsorbed onto the xerogel glass. Cells on xerogel glass without BMP were more differentiated than cells grown on plastic with BMP, thereby demonstrating the additive effect of a bioactive substrate and BMP on osteoblastic cell differentiation. These data indicate that xerogel glass effects differentiation of cells with osteogenic potential and that it can serve as delivery vehicle for BMP.
GARCIA AJ,
DUCHEYNE
P, BOETTIGER D
THE EFFECT OF SURFACE REACTION STAGE ON FIBRONECTIN-MEDIATED ADHESION
OF OSTEOBLAST-LIKE CELLS TO BIOACTIVE GLASS.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 40(1):48-56, 1998 APR.
Bioactive glasses and ceramics enhance bone formation and bond directly to bone, and have emerged as promising substrates for bone tissue engineering applications. Bone bioactivity involves physicochemical surface reactions and cellular events, including cell attachment to adsorbed extracellular matrix proteins. The effects of fibronectin (Fn) adsorption and glass surface reaction stage on the attachment of osteoblast-like cells (ROS 17/2.8) to bioactive glass were analyzed. Bioactive glass disks were pretreated in a simulated physiologic solution to produce three reaction layers: unreacted glass (BG0), amorphous calcium phosphate (BG1d), and carbonated hydroxyapatite (BG7d). Synthetic hydroxyapatite (sHA) and nonreactive borosilicate glass (CG) were used as controls. A spinning disk device which applied a linear range of forces to attached cells while maintaining uniform chemical conditions at the interface was used to quantify cell adhesion. The number of adherent cells decreased in a sigmoidal fashion with applied force, and the resulting detachment profile provided measurements of adhesion strength. For the same amount of adsorbed Fn, cell adhesion was higher on surface-reacted bioactive glasses (BG1d and BG7d) than on BG0, CG, and sHA. For all substrates, cell attachment was primarily mediated by the RGD binding site of Fn, as demonstrated by blocking experiments with antibodies and RGD peptides. Cell adhesion strength increased linearly with adsorbed Fn surface density. Analysis of this fundamental relationship revealed that improved adhesion to reacted bioactive glasses resulted from enhanced cell receptor-Fn interactions, suggesting substrate-dependent conformational changes in the adsorbed Fn.
Using one parametric variations in solution composition this paper
documents
that the surface reactions on bioactive glass (BG) 45S5 are exquisitely
dependent upon the modelling conditions. The solutions used were 0.05 M
tris hydroxymethyl aminomethane/HCl (tris buffer); tris buffer
complemented
with plasma electrolyte and/or serum, and serum. The reacted
surfaces
were analyzed using Fourier transform infrared (FTIR), scanning
electron
microscopy (SEM) with energy dispersive X-ray analysis (EDXA), and
Rutherford
backscattering spectroscopy (RBS). Post-immersion solutions were
analyzed
for changes in Ca and PO4 concentrations.
After a short immersion (3 h) a crystalline, carbonated hydroxyapatite
(c-HA) layer formed only in tris. Reaction surfaces of different
structure,
morphology and composition were observed after various short and longer
term immersions in all other solutions. They comprised two layers with
the layer in contact with the bulk consisting mainly of Si; the outer
layer
was composed of Si, Ca and P, was amorphous and had a Ca/P ratio of
about
1. Serum proteins adsorbed on the BG surfaces at the early stages of
the
solution-mediated BG reactions. Formation of a crystalline c-HA layer
was
delayed up to three or more days in solution with plasma ions. In the
presence
of serum, only amorphous surfaces composed of Si, Ca and P were
observed
for any time up to seven days of immersion. The present data
suggest
that serum proteins adsorb in tandem with the occurrence of
solution-mediated
reactions leading to formation of a silica-gel. Amorphous CaP-phases
accumulate
in the Si-rich matrix. Furthermore, the present data in conjuction with
the data published before1, suggest that physicochemical and
cell-mediated
reactions occur in parallel to form the glass-tissue interfacial layer.
EL-GHANNAM A, DUCHEYNE P, SHAPIRO IM.
BIOACTIVE MATERIAL TEMPLATE FOR IN VITRO SYNTHESIS OF BONE.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 29(3):359-70, 1995 MAR.
We describe the synthesis of a new, porous, modified bioactive glass for use as a template for bone formation in vitro. The porosity of the glass was 36.4%; the pore size ranged from 10-160 mm, and there was no incipient devitrification. Prior to seeding the glass with cells, it was necessary to condition the disks. Optimum conditioning was achieved by immersing the templates in a tris buffer at pH 6.8 for 48 h and then treating the glass with tissue culture medium for 1 h at 37 degrees C. The conditioned glass disks were seeded with 10(6) neonatal rat calvaria osteoblast-like cells; cells on the substrate were maintained in culture for 3-7 days. To prevent pH shifts due to corrosion of the conditioned glass, the medium:glass ratio was maintained at 90 ml/g. We found that the templates were rapidly invaded by cells which maintained the osteoblast phenotype; thus, they exhibited high alkaline phosphatase activity and synthesized type I collagen and osteocalcin. SEM-EDAX showed that the cells elaborated substantial amounts of extracellular matrix and a bonelike tissue was substantial amounts of extracellular matrix and a bonelike tissue was present throughout the entire template thickness. FTIR analysis of material formed in the glass indicated that the mineral phase was a biologic hydroxyapatite. Controls (cells without substrate and substrate without cells) exhibited none of these features. Results of the study suggest that this porous glass can function as a template for generating bone in vitro.
Using in vitro synthesized bone tissue with cells aspirated from the patient's marrow is an appealing idea to avoid the profound limitations of biological and synthetic grafts. Procedures to synthesize bone tissue in vitro primarily relied on seeding various substrates with cells that have osteogenic capacity in culture. It should be noted that in an in vitro system, osteoprogenitor cells, as well as bone cells themselves can rapidly change their phenotype, hence the substrate needs to promote the expression of the bone cell phenotype. Furthermore, it needs to provide a template for bone deposition while gradually resorbing once bone tissue has been laid down. This paper presents initial evidence that bioactive glass, a synthetic material with documented extensive bone bioactivity properties, represents a material that optimally combines the requirements of the ideal template for in vitro synthesis of bone tissue. When made in porous form, and conditioned to develop a bone-like surface prior to being seeded with pluripotential cells capable of expressing the osteoblastic phenotype, these templates lead to expeditious and abundant in vitro synthesis of extracellular matrix with most important characteristics of bone tissue. [References: 24]
DUCHEYNE P.
BIOACTIVE CERAMICS [EDITORIAL].
JOURNAL OF BONE & JOINT SURGERY - BRITISH VOLUME. 76(6):861-2,
1994 NOV.
SCHEPERS EJ, DUCHEYNE P, BARBIER L, SCHEPERS S
BIOACTIVE GLASS PARTICLES OF NARROW SIZE RANGE: A NEW MATERIAL FOR
THE REPAIR OF BONE DEFECTS.
IMPLANT DENTISTRY. 2(3):151-6, 1993 FALL.
A clinical trial to treat dental osseous lesions with bioactive glass granules of narrow size range (300 to 360 microns) has been conducted since early spring 1990. This study followed an animal study in which the osseous tissue repair properties of bioactive glass granules of narrow size range and hydroxyapatite granules were compared for up to 2 years of implantation. The therapeutic response to the bioactive glass material exceeded the response to the hydroxyapatite as evidenced by very extensive osteoconduction, as well as the capacity to cause differentiation of steoprogenitor cells to osteoblasts. The clinical study was started by virtue of the bioactive glass granules of narrow size range eliciting expeditious bone tissue formation throughout a defect. In this clinical study 87 patients and 106 defects were treated. The indications selected were apical resection areas, cystic defects, extraction sites, and defects of the alveolar ridge due to surgery or resorption. After insertion, the particles remained well in place and only small changes in the contours of the restored defects were seen, at the most up to 2 months postinsertion. At 3 months the application sites had fully solidified. Radiographic analysis indicated that the material integrated into the bone tissue, and at 6 months any difference between glass particles and bone tissue had nearly disappeared. The few initial cases with limited clinical results were caused by factors unrelated to the glass granules, mostly the surgical technique. By adapting the surgical technique, no unfavorable clinical results were subsequently experienced.(ABSTRACT TRUNCATED AT 250 WORDS)
HEALY KE, DUCHEYNE P.
THE MECHANISMS OF PASSIVE DISSOLUTION OF TITANIUM IN A MODEL
PHYSIOLOGICAL
ENVIRONMENT.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 26(3):319-38, 1992 MAR.
The surface chemistry, oxidation, and disolution kinetics of titanium were measured to establish the mechanisms of passive dissolution in physiological environments. Titanium thin films were immersed in 8.0 mM ethylenediamine-tetraacetic acid in simulated interstitial electrolyte (EDTA/SIE) and maintained at 37 degrees C, 10% O2, 5% CO2 and 7.2 pH for periods of time up to 3200 h (133 days). Two immersion schemes were employed: the integral sequentially determined the titanium released into a solution of accumulated dissolution products; and the differential continuously replenished the test solution. The solutions were analyzed for titanium by electrothermal atomic absorption spectrometry (EAAS), and the sample surfaces were analyzed by Auger electron spectroscopy (AES) and x-ray photoelectron spectroscopy (XPS) to determine oxide composition, stoichiometry, and thickness. Prior to immersion two types of hydroxyl (OH) groups were distinguished on the TiO2 surface. Upon immersion, the chemistry of the surface changed as a function of immersion: the pressure of OH groups increased and P (nonelemental) was detected at the surface. The dissolution kinetics obeyed a two-phase logarithmic model, where the transition between phases occurred simultaneously with the adsorption of the P-containing species. The dissolution kinetics depended on surface reactions, electric field strength, and molecular diffusion. These mechanisms explain the observed dependence of dissolution kinetics on the properties of the surface oxide and solution ligands.
KOHN DH, DUCHEYNE P.
A PARAMETRIC STUDY OF THE FACTORS AFFECTING THE FATIGUE STRENGTH OF
POROUS COATED TI-6A1-4V IMPLANT ALLOY.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 24(11):1483-501, 1990 NOV.
The high cycle fatigue strength of porous coated Ti-6A1-4V is approximately 75% less than the fatigue strength of uncoated Ti-6A1-4V. This study separates the effects of three parameters thought to be responsible for this reduction: interfacial geometry, microstructure, and surface alterations brought about by sintering. To achieve the goal of one parameter variations, hydrogen-alloying treatments, which refined the lamellar microstructure of beta-annealed and porous coated Ti-6A1-4V, were formulated. The fatigue strength of smooth-surfaced Ti-6A1-4V subjected to hydrogen-alloying treatments is 643-669 MPa, significantly greater than the fatigue strength of beta-annealed Ti-6A1-4V (497 MPa) and also greater than the fatigue strength of pre-annealed, equiaxed Ti-6A1-4V (590 MPa). The fatigue strength of porous coated Ti-6A1-4V, however, is independent of microstructure. This leads to the conclusion that the notch effect of the surface porosity does not allow the material to take advantage of the superior fatigue crack initiation resistance of a refined alpha-grain size. Thus, sinternecks acts as initiated microcracks and fatigue of porous coated Ti-6A1-4V is propagation controlled.
DUCHEYNE P
BIOCERAMICS: MATERIAL CHARACTERISTICS VERSUS IN VIVO BEHAVIOR
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH 21(A2 Suppl):219-36, 1987
Aug.
DUCHEYNE P; RADIN S; KING L
THE EFFECT OF CALCIUM-PHOSPHATE CERAMIC COMPOSITION AND STRUCTURE ON
INVITRO BEHAVIOR .1. DISSOLUTION
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 27(1): 25-34 1993 JAN
Synthetic calcium phosphate ceramic (CPC) surfaces can be transformed to a biological apatite through a sequence of reactions which include dissolution, precipitation, and ion exchange. By virtue of the reactions being material-dependent, it is important to determine parametric rate effects. In this study we focused on the effect of stoichiometry and crystal structure of CPCs on the dissolution kinetics. Monophase, biphase, and multiphase CPCs with a Ca/P ratio equal to or greater than 1.5 were studied. The experiments were performed in a calcium and phosphate-free Tris buffer solution at pH 7.3. The dissolution behavior of the CPCs studied was found to vary over a wide range. The dissolution rate of the monophase CPCs increased in the order of stoichiometric hydroxyapatite, calcium deficient hydroxyapatite, oxyhydroxyapatite, beta-tricalcium phosphate, alpha-tricalcium phosphate, and tetracalcium phosphate. Dissolution of biphase and multiphase CPCs increased prorated the concentration of more soluble component. PT Article
RADIN SR; DUCHEYNE P
THE EFFECT OF CALCIUM-PHOSPHATE CERAMIC COMPOSITION AND STRUCTURE ON
INVITRO BEHAVIOR .2. PRECIPITATION
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 27(1): 35-45 1993 JAN
The formation of a biologically equivalent carbonate-containing apatite on the surface of synthetic calcium phosphate ceramics (CPC) may be an important step leading to bonding with bone. Reactions of several single phases CPCs upon immersion into a simulated physiologic solution (SPS) with an electrolyte composition of human plasma were determined. The CPCs covered a wide range of solution stabilities from low-soluble hydroxyapatites (HA) to metastable tricalcium phosphates (TCP) and tetracalcium phosphate (TTCP). Changes in chemical compositions of SPS and infrared spectral features after CPC immersion were analyzed. New phase formation was observed on all the CPCs. However, kinetics, compositions, and structures of the new phases were significantly different. The studied CPCs can be characterized by the time to new phase formation in vitro; the minimum time for measurable precipitate formation was found to increase in the order: not-well-crystallized HAs ) well-crystallized HAs ) alpha-TCP, TTCP ) beta-TCP. Among the CPCs only not-well-crystallized HAs led to immediate new phase formation. The metastable CPCs, beta-TCP, alpha-TCP, and TTCP required an induction time during which dissolution occurred. Beta-TCP showed the longest induction time and the lowest lattice ion uptake rate of all the CPCs tested. Only the not-well-crystallized HAs elicited immediate formation of carbonated HA. The well-crystallized HAs and beta-TCP did not elicit carbonated apatite formation within the time frame of the experiment. Instead, intermediate phases were formed. On alpha-TCP amorphous calcium phosphate (ACP) with a relatively low carbonate content was formed. TTCP was found to transform extensively to poorly crystallized carbonated apatite after 2 days of immersion.
DUCHEYNE P. HENCH LL. KAGAN A 2D. MARTENS M.
BURSENS
A. MULIER JC.
EFFECT OF HYDROXYAPATITE IMPREGNATION ON SKELETAL BONDING OF POROUS
COATED IMPLANTS.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 14(3):225-37, 1980 MAY.
Skeletal fixation of permanent implants by new methods such as fixation by mechanical interlocking of bone with porous prosthetic coatings or chemical bonding with bioactive materials shows growing potential. This paper reports on the resulting skeletal fixation of a combined porous and bioactive material. Metal plugs with a porous metal fiber coating impregnated with hydroxyapatite were implanted for 2, 4, and 12 weeks and were compared to the parent porous, nonbioactive, metal fiber material. Statistical analysis of the interfacial failure shear stress, as obtained by mechanical testing, shows there is a marked influence of hydroxyapatite impregnation on the rate of bone ingrowth and the strength of the interfacial bond the few weeks following surgery. Microscopical examination reveals that the apparent stimulation of bone ingrowth into the surface pores of the implant is the reason for the increased rate of bond formation. The results are of particular clinical interest: with an increased rate of bone ingrowth, weight bearing might be allowed much earlier, thus reducing the recuperation period.