[Attaching single- and multi-unit fixed dental prostheses]. / Bevestiging van kronen en bruggen.

A single- or multi-unit fixed dental prosthesis can be attached to the abutment teeth through mechanical retention and gap sealing or by adhesion. For sealing the gap, water-soluble cements are appropriate, such as zinc phosphate, polycarboxylate, and (resin-modified) glasionomer cement. Attachment through adhesion can be performed with composite cement. If the hard tooth tissue is prepared adequately, composite cement provides sufficient adhesion, but self-adhesive composite cement is now also available. For the adhesion of the composite cement to the restorative materials of the single- or multi-unit fixed dental prosthesis, surface sandblasting, silanizing, and tin coating and the application of a metal primer or chemically active composite are available. Cementing a single- or multi-unit dental prosthesis involves 3 phases: 1. Cleansing the single- or multi-unit dental prosthesis and the abutment tooth/teeth; 2. Preparing the hard tooth tissue, mixing the cement and placing the single- or multi-unit dental prosthesis; 3. Removing the excess cement.

Ceramic composites as matrices and scaffolds for drug delivery in tissue engineering.

Ceramic composites and scaffolds are popular implant materials in the field of dentistry, orthopedics and plastic surgery. For bone tissue engineering especially CaP-ceramics or cements and bioactive glass are suitable implant materials due to their osteoconductive properties. In this review the applicability of these ceramics but also of ceramic/polymer composites for bone tissue engineering is discussed, and in particular their use as drug delivery systems. Overall, the high density and slow biodegradability of ceramics is not beneficial for tissue engineering purposes. To address these issues, macroporosity can be introduced often in combination with osteoinductive growth factors and cells. Ceramics are good carriers for drugs, in which release patterns are strongly dependent on the chemical consistency of the ceramic, type of drug and drug loading. Biodegradable polymers like polylactic acid, gelatin or chitosan are used as matrices for ceramic particles or as adjuvant to calcium phosphate cements. The use of these polymers can introduce a tailored biodegradation/drug release to the ceramic material.

The in vitro behavior of as-prepared and pre-immersed RF-sputtered calcium phosphate thin films in a rat bone marrow cell model.

In this paper we focus on the behavior of radio frequency (RF)-sputtered calcium phosphate (CaP) thin films in a rat bone marrow (RBM) cell model. Two issues are addressed. Firstly, we benchmarked the in vitro cell behavior of these CaP coatings by comparing their proliferation, differentiation and mineralization behavior and the structure of the formed interface to similar coatings of alumina and titania. We found that the CaP coatings showed reduced proliferation, enhanced early differentiation and enhanced activity of mature osteoblasts compared to the alumina coatings. Enhanced production of mineralized extracellular matrix (ECM) was seen for both CaP and titania. Two types of CaP precipitates could be observed, one directly bonded CaP layer at the coating interface and one of globular accretions associated with the ECM. The directly bonded layer was not observed on the alumina coatings. Further, no thin film effects were found. Secondly, the effect of pre-immersion of the CaP coatings in SBF2 was explored. We found that the early formation of a directly bonded CaP layer is obstructed by the absence of CaP nuclei. After approximately 8 days, cell activity induces the nucleation of CaP crystals on both the surface and the ECM, and growth is enhanced. By initially providing these coatings with CaP crystals, growth of the directly bonded CaP layer is immediate. Hence, the formation of the interfacial CaP layer and the matrix-associated CaP accretions can effectively be decoupled.

Preparation and characterization of RF magnetron sputtered calcium pyrophosphate coatings.

CaP ceramic has been widely used as coating on metals in orthopedics and oral dentistry. Variations in CaP composition can lead to different dissolution/precipitation behavior and may also affect the bone response. In the present study calcium pyrophosphate and hydroxylapatite coatings were successfully prepared by RF magnetron sputtering deposition. The phase composition, morphological properties, and the dissolution in SBF were characterized by using XRD, FTIR, EDS, SEM, and spectrophotometry. The results showed that all the sputtered coatings were amorphous and changed into a crystal structure after IR-radiation. The temperature for the crystallization of the amorphous coatings is lower for the hydroxylapatite coating (550 degrees C), compared to the calcium pyrophosphate coating (650 degrees C). All sputtered amorphous coatings were instable in SBF and dissolved partially within 4 wks of incubation. The heat-treated coatings appeared to be stable after incubation. These results showed that magnetron sputtering of calcium pyrophosphate coating is a promising method for forming a biocompatible ceramic coating.

Subcutaneous evaluation of RF magnetron-sputtered calcium pyrophosphate and hydroxylapatite-coated Ti implants.

The in vivo behavior of infrared-heated, RF magnetron-sputtered hydroxylapatite (HA) and calcium pyrophosphate (DCPP) coated titanium discs was investigated. The discs were implanted subcutaneously in the back of six goats for 2, 4, 8 and 12 weeks. At the end of the study, coated discs were removed and examined on their physicochemical properties by X-ray diffraction (XRD) and scanning electron microscopy (SEM), including energy dispersive spectroscopy (EDS). Also, implants were prepared for light microscopical evaluation of the tissue response. The results showed that heat-treated HA coatings showed a stable behavior, i.e. no changes in the XRD pattern occurred during implantation. Also, no dissolution of the coating was observed by SEM. EDS revealed that the Ca/P ratio of the HA coatings remained stable during implantation. In contrast, heat-treated DCPP coatings showed a compositional change into apatite and tricalcium phosphate (TCP) during implantation. This was confirmed by the SEM and EDS analysis. The Ca/P ratio of the DCPP coatings changed from 0.8 to 1.52 during implantation. Finally, histology showed that both heat-treated HA and DCPP coatings showed no adverse tissue response, as characterized by the presence of thin, dense fibrous tissue capsule. Consequently, it can be concluded that 2 mum thick heat-treated, RF magnetron-sputtered HA and DCPP coatings are of sufficient thickness to withstand dissolution during 12 weeks of implantation in a subcutaneous location in goats. In addition, both coatings showed a biocompatible tissue behavior. Further, heat-treated DCPP coatings revealed a gradual compositional change into apatite and TCP.

Closing capacity of cranial bone defects using porous calcium phosphate cement implants in a rabbit animal model.

Calcium phosphate (Ca-P) cement is a well established material for bone repair. The bone biological properties of Ca-P cement can even be further improved by creating porosity in the material. The current study aimed on the evaluation of the osteoconductive behavior of porous Ca-P cement. Therefore, circular defects (6, 9, and 15 mm in diameter) were created in the cranium of 3 months old rabbits and filled with porous Ca-P cement implants. The total porosity of implants was calculated to be 71, 74 and 74% respectively and the average pore diameter was 150 microm. In addition, empty control defects were prepared. After 12 weeks implantation time the animals were sacrificed and radiographic, histological, and histomorphometrical evaluation was performed. The Critical Size Defect (CSD) of this species at this location for an implantation time of 12 weeks was confirmed to be 15 mm. Bone was observed to be present over and through almost all porous Ca-P cement implants. Only, in one out of eight animals with a 15 mm implant complete bone bridging of the defect did not occur. The size of the defect was found not to affect the total percentage of bone formation in the cement; (17 +/- 7)%, (18 +/- 6)% and (17 +/- 3)% for respectively 6, 9, and 15 mm diameter implants. We concluded that porous Ca-P cement is an excellent osteoconductive material in non weight bearing situations and complete bridging of a critical sized skull defect occurs in this rabbit model after 12 weeks of implantation.

Growth behavior of rat bone marrow cells on RF magnetron sputtered hydroxyapatite and dicalcium pyrophosphate coatings.

The aim of this study was to evaluate the osteogenic properties of magnetron sputtered dicalcium pyrophaosphate (DCPP) and hydroxylapatite (HA) coatings. Therefore, DCPP and HA coatings were deposited on grit-blasted titanium discs. The substrates were used as-prepared or received an additional heat treatment which changed the amorphous coating structure to a crystalline structure. Subsequently, rat bone marrow stromal cells were cultured for 1-24 days on the various substrates. DNA and alkaline phosphatase activity was determined after 1, 3, 5, 8, and 12 days of incubation. Osteocalcin expression was evaluated after 8, 12, 16, and 24 days of incubation. Scanning electron microscopical analysis of cell morphology and coating characteristics was done after 8 and 16 days of incubation. All assays were done in duplicate and in each assay all specimens were present in fourfold. Results demonstrated that the cells did not proliferate and differentiate on all amorphous coatings. SEM revealed that the amorphous coatings showed significant dissolution. On the crystalline DCPP and HA coatings an increase in DNA and alkaline phosphatase activity was seen starting at day 8 of incubation. Osteocalcin expression on the crystalline coatings started to increase at day 16 of incubation. SEM showed that the growth and differentiation of the cells was associated with extensive collagen fiber formation and surface mineralization in the form of globular accretions. Further, statistical testing revealed that proliferation and differentiation of the rat bone marrow stromal cells started significantly earlier on the crystalline HA coatings than that on the crystalline DCPP coatings. These results demonstrate that the rat bone marrow stromal cells proliferated and differentiated only on crystalline magnetron sputtered DCPP as well as HA coatings, which warrants the further in vivo analysis of the bone healing supporting properties of these coatings.

The influence of the crystallinity of electrostatic spray deposition-derived coatings on osteoblast-like cell behavior, in vitro.

This article describes the influence of the crystallinity of carbonate apatite (CA) coatings on osteoblast-like cell behavior. Porous CA coatings were produced with electrostatic spray deposition (ESD), and subsequently, received heat treatments of 400, 500, or 700 degrees C to induce various coating crystallinities. As a result, an amorphous calcium phosphate (ACP), a crystalline CA (CCA), and a crystalline carbonated hydroxyapatite (CHA) structure were formed, respectively. Uncoated titanium substrates served as the control group. After seeding rat osteoblast-like cells, the initial cell attachment was similar between the groups, and approached 100% after 6 h. Between the various coatings, no differences were observed for proliferation, differentiation, or mineralization. However, proliferation of the osteoblast-like cells was lower on all coated substrates after longer culture periods, compared to the uncoated substrates, while at the same time differentiation was stimulated. Furthermore, after 8 and 16 days of incubation, scanning electron microscopy showed more signs of mineralization on coated substrates, compared to the uncoated substrates. In conclusion, porous ESD-derived CA coatings have a positive effect on the in vitro differentiation of osteoblast-like cells, compared to uncoated, as-machined titanium. However, this effect is not further enhanced by the degree of crystallinity of the ESD-derived CA coatings.

Mechanical properties of porous, electrosprayed calcium phosphate coatings.

Mechanical properties of calcium phosphate coatings (CaP), deposited using the electrostatic spray deposition (ESD) technique, have been characterized using a range of analytical techniques, including tensile testing (ASTM C633), fatigue testing (ASTM E855), and scratch testing using blunt and sharp scratch styli. Moreover, a simple explantation procedure was successfully introduced using ESD-coated, threaded dental implants to characterize the mechanical performance of CaP coatings qualitatively under conditions that mimic clinical situations as close as possible. Generally, all analysis techniques revealed that ESD coatings need to be crystallized in order to ensure interfacial adhesion to the substrate and sufficient mechanical strength of the superficial reticular structure. Crystalline carbonated hydroxyapatite coatings (CHA, heat-treated at 700 degrees C) were resistant to fatigue as well as to plastic ploughing deformation by means of various scratch styli, and the fragile surface structure of ESD coatings was maintained to a large extent after unscrewing CHA-coated dental implants from femoral condyles of goat cadavers. From these experiments, it was concluded that interfacial adhesion of crystalline CHA ESD coatings to the titanium substrate was sufficient, but that mechanical strength of the superficial architecture of ESD coatings need to be optimized for applications where high shear and compressive stresses are imposed onto the rather fragile coating surface of reticular ESD morphologies.

Differentiation ability of rat postnatal dental pulp cells in vitro.

The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats. Immunochemistry by stem cell marker STRO-1 proved the existence of stem cells or progenitors in the isolated cell population. The dissociated cells were then cultured both on smooth surfaces and on three-dimensional (3-D) scaffold materials in medium supplemented with beta-glycerophosphate, dexamethasone, and L-ascorbic acid. Cultures were analyzed by light and scanning electron microscopy and, on proliferation, alkaline phosphatase activity and calcium content were determined and the polymerase chain reaction was performed for dentin sialophosphoprotein, osteocalcin, and collagen type I. These cells showed the ability to differentiate into odontoblast-like cells and produced calcified nodules, which had components similar to dentin. In addition, we found that the "odontogenic" properties of the isolated cells were supported by three-dimensional calcium phosphate and titanium scaffolds equally well.

Osteoclastic resorption of calcium phosphate coatings applied with electrostatic spray deposition (ESD), in vitro.

Calcium phosphate (CaP) coatings have been applied on titanium implants to improve the bioactivity in order to favor the initial bone healing response. Recently, a new technique has been developed to apply CaP coatings: electrostatic spray deposition (ESD). Although ESD-derived coatings have several benefits, it is not known whether they are degradable. This study was designed to examine the cell-mediated degradation of two ESD-derived coatings with different chemical compositions, that is, beta-tricalcium phosphate (beta-TCP) and carbonate apatite (CA). First, coatings were deposited and analyzed physiochemically. Subsequently, rat bone marrow-derived osteoclastlike cells were seeded on the coatings, and analyzed with osteoclast-specific markers, scanning electron microscopy, and transmission electron microscopy. Results showed that both coatings exhibited porous morphologies, with an average pore size of less than 1 microm (beta-TCP), or larger than 1 microm (CA). After heat treatment, both coatings were crystalline in structure. The Ca/P ratios were 1.4 to 1.5 for the beta-TCP coating, and 1.8 to 2.0 for the CA coating. After 8 and 12 days of culture, multinucleated osteoclastlike cells were observed on both coatings. The osteoclast phenotype was confirmed by tartrate resistant acid phosphatase (TRAP) staining, and immunostaining against the calcitonin receptor. Using scanning electron microscopy, numerous resorption lacunae were observed in both coatings. Finally, transmission electron microscopy of TRAP-positive cells confirmed the osteoclastlike aspect of the cells revealing multiple nuclei and a ruffled border. In conclusion, CaP coatings produced with the ESD process can be degraded by osteoclasts.

A new method to produce macropores in calcium phosphate cements.

A new way to create macropores in calcium phosphate cements has been developed. The method consists in adding NaHCO3 to the starting cement powder (Biocement D) and using two different liquids: first a basic liquid to form the paste and later an acid liquid to obtain CO2 bubbles. Mercury intrusion measurements showed a dramatic increase both in macropores with an average size of 100 m and in the total porosity (even higher than 50% with respect to the Biocement D). This method does not change in any significant way the final reaction products of the starting material after being soaked 3 days in Ringer solution. Only, due to the increase of the porosity. the compressive strength of the porous cement decreases significantly.

Soft-tissue response to injectable calcium phosphate cements.

In this study, the soft tissue reaction to two newly developed injectable calcium phosphate bone cements (cement D and W) was evaluated after implantation in the back of goats. For one of the cements (cement D) the tissue reaction was also investigated after varying the concentration of accelerator Na(2)HPO(4) in the cement liquid (resulting in cement D1 and D2). Eight healthy mature female Saanen goats were used. The cement was applied 10min after mixing while it was still moldable and plastic. The material was given a standardized cylindrical shape. Thirty-two implants of each cement formulation were inserted and left in place for 1, 2, 4, and 8weeks. At the end of the study, eight specimens of each material and healing period were available for further analysis. Two specimens were used for X-ray diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) and six specimens were used for light microscopical evaluation. XRD and FTIR showed that the cements did set as microcrystalline carbonate apatite with the disappearance of monetite from the cements during implantation. Histological analysis showed that after 8weeks of implantation around all materials a thin soft-tissue capsule was formed (thickness ranging from 5 to 15 cell layers) with almost complete absence of inflammatory cells. Only in some specimens a slightly higher inflammatory reaction was observed. This was due to cement surface defects and a zone of dispersed particles near the cement-soft tissue interface. There was almost no resorption of the material after 8 weeks of implantation. In a few 4 and 8weeks samples, small areas of calcification were found in the fibrous capsule surrounding the implants. On the basis of our observations, we conclude that the tested cements were biocompatible and can be used next to soft tissue.

Initial deposition of calcium phosphate ceramic on polyethylene and polydimethylsiloxane by rf magnetron sputtering deposition: the interface chemistry.

Calcium phosphate (CaP) coatings are well known for their bioactive nature. CaP coated polymeric materials can be used as implant material. For this, a strong adhesion between the coating and substrate is necessary. Because the chemical structure of the interface plays an important role in the coating adhesion, we studied the interface between CaP and the polymers polyethylene (PE) and polydimethylsiloxane (PDMS/silicone rubber). Both untreated and plasma pretreated polymers were used. On PE, a low Ca/P ratio nearby the interface and a high amount of C-O bonds were found on both untreated and plasma pretreated PE. This is the result of phosphate-like groups that are able to bind to the carbon of the PE. PDMS reacts towards the plasma pretreatment by losing CH(3) side groups. Compared to PE, a low amount of C-O bonds is found nearby the interface. Besides, a low Ca/P ratio is found nearby the interface. This is the result of phosphate groups that connect to Si atoms of the PDMS, thereby replacing the CH(3) side groups. The bombardment by negatively charged oxygen ions that are accelerated from the target during the deposition process makes the chemical interaction between the coating and the substrates possible.

Influence of precursor solution parameters on chemical properties of calcium phosphate coatings prepared using Electrostatic Spray Deposition (ESD).

A novel coating technique, referred to as Electrostatic Spray Deposition (ESD), was used to deposit calcium phosphate (CaP) coatings with a variety of chemical properties. The relationship between the composition of the precursor solutions and the crystal and molecular structure of the deposited coatings was investigated by means of X-ray diffraction (XRD), Fourier-Transform Infrared Spectroscopy (FTIR) and Energy Dispersive Spectroscopy (EDS). It was shown that the relative Ca/P ratio in the precursor solution, the absolute precursor concentration, the acidity of the precursor solution and the type of Ca-precursor strongly influenced the chemical nature of the deposited CaP coatings. Various crystal phases and phase mixtures were obtained, such as carbonate apatite, beta-TCP, Mg-substituted whitlockite, monetite, beta/gamma-pyrophosphate, and calcite. It was shown that carbonate plays an essential role in the chemical mechanism of coating formation. Carbonate is formed due to a decomposition reaction of organic solvents. Depending on deposition conditions, carbonate anions (a) react with acidic phosphate groups, (b) are incorporated into apatitic calcium phosphate phases, and (c) react with excessive Ca(2+) cations in case of phosphate-deficient precursor solutions.

Enhancement of initial stability of press-fit femoral stems using injectable calcium phosphate cement: an in vitro study in dog bones.

In this in vitro study we evaluated the initial stability of cementless femoral stems using an injectable calcium phosphate (Ca-P) cement. The cement was not used to form a cement mantle as is routinely done in PMMA cemented prostheses but functioned as an additive to fill the small gaps that exist between a press-fit placed titanium plasma sprayed implant and the bone bed. Six pair of Beagle femora were used in this study. In a random fashion, one femur of each pair was used for placement of a prosthesis without Ca-P cement, the contralateral was used for press-fit placement after injection of the calcium phosphate cement into the intramedullary canal. The reconstructions were placed in a MTS testing machine, tilted 15 degrees in varsus and 15 degrees of endorotation to obtain a physiological load on the femoral head. The load was applied stepwise from zero to a maximum of 100, 250 and 400 N, respectively. At each loading step the load was applied dynamically at a frequency of 1 Hz for 30 min. Between the loading steps, the load was removed for 10 min to allow elastic recovery. The stability of the stems was determined at each loading step with roentgen-stereophotogrammetric analysis. Results showed that with the prostheses without Ca-P cement the most important displacements were movement into varus (max. 818 microm under 400 N) and subsidence (max. 587 microm under 400 N). The displacements showed large variation. After unloading some elastic recovery occurred. In the specimens with Ca-P cement, displacements were negligible. As determined by an F-test the variations found were significantly smaller for the press-fit+Ca-P cement relative to the press-fit prosthesis at all loading steps (p<0.05). A paired t-test revealed significant differences in the mentioned displacements between the press-fit- and press-fit+Ca-P cement prosthesis at a loading with 400 N (P<0.05). On the basis of these results we conclude that the use of Ca-P cement increases the initial stability of press-fit inserted plasma-sprayed femoral prostheses and corrects for the high variability in displacements found with press-fit insertion of these femoral hip prostheses.

Electrostatic spray deposition (ESD) of calcium phosphate coatings, an in vitro study with osteoblast-like cells.

Electrostatic spray deposition (ESD) is a recently developed technique to deposit a calcium phosphate (CaP) coating upon substrates. With this technique, an organic solvent containing calcium and phosphate is pumped through a nozzle. Between the nozzle and substrate a high voltage is applied. As a consequence, droplets coming out the nozzle disperse into a spray, and this spray is deposited upon the substrate. When the solvent has evaporated, a coating is formed on the substrate. ESD allows for a variation in coating composition and morphology. Titanium alloy (TiAl6V4) substrates were coated with a CaP layer using two different methods; radio frequency magnetron sputtering, and ESD. These surfaces were characterized with X-ray diffraction, Fourier transform infrared spectroscopy, an universal surface tester, scanning electron microscopy, and energy dispersive spectrometry. Subsequently, bone marrow cells were isolated from rat femora and cultured 1, 4, 8, 14 and 16 days. Cell proliferation, alkaline phosphatase activity, and osteocalcin concentration were assayed. RT-PCR was done for collagen type I and osteocalcin. SEM was also performed to observe cellular behaviour during culture. Two separate runs of the experiment were performed. In the first run, osteoblast-like cells on both CaP coatings showed similar results in all assays. In the second run, proliferation and osteogenic expression had increased on ESD coatings. On basis of these results, we conclude that the novel ESD coating behaved similar to, or even better than the known RF magnetron sputter coating. Thus, ESD could be a valid addition to already existing CaP coating processes.

In vivo dissolution behavior of various RF magnetron-sputtered Ca-P coatings on roughened titanium implants.

RF magnetron sputter deposition was used to produce 0.1, 1.0 and 4.0 microm thick Ca-P coatings on TiO(2)-blasted titanium discs. Half of the as-sputtered coated specimens were subjected to an additional infrared heat treatment for 30s at 425-475 degrees C. X-ray diffraction demonstrated that infrared radiation changed the amorphous 4 microm sputtered coatings into an amorphous-crystalline structure, while the amorphous 0.1 and 1 microm changed in a crystalline apatite structure with the presents of tetracalciumphosphate as a second phase. Scanning electron microscopically examination of the sputtered coatings revealed that annealing of the 4 microm thick coatings resulted in the appearance of small cracks. Subsequently, the discs were implanted subcutaneous into the back of rabbits. After 1, 4, 8 and 12 weeks of implantation, the implants were retrieved and prepared for histological and physicochemical evaluation. Histological evaluation revealed that the tissue response to all coated implants was very uniform. A very thin connective tissue capsule surrounded all implants. The capsule was usually free of inflammatory cells. At the interface, there was a close contact between the capsule and implant surface and no inflammatory cells were seen. Physicochemical evaluation showed that the 0.1 and 1 microm thick amorphous coatings had disappeared within 1 week of implantation. On the other hand, the 4 microm thick amorphous phase disappeared during the implantation periods, which was followed by the precipitation of a crystalline carbonate apatite. Further, at all implantation periods the heat-treated 1 and 4 microm thick coatings could be detected. Occasionally, a granular precipitate was deposited on the heat-treated 4 microm thick coating. Fourier transform infrared spectroscopy showed the formation of carbonate apatite (CO(3)-AP) on the 4 microm thick amorphous coating and on the heat-treated specimens. On basis of our findings, we conclude that 1 microm thick heat-treated Ca-P sputter coating on roughened titanium implants appear to be of sufficient thickness to show bioactive properties, under in vivo conditions.

Transforming growth factor-beta1 incorporation in an alpha-tricalcium phosphate/dicalcium phosphate dihydrate/tetracalcium phosphate monoxide cement: release characteristics and physicochemical properties.

The osteoconductive properties of calcium phosphate cements (CPCs) may be improved by the addition of growth factors, such as recombinant human transforming growth factor-beta1 (rhTGF-beta1). Previously we have shown that rhTGF-beta1 was released from cement enriched with rhTGF-beta1 and subsequently stimulated the differentiation of pre-osteoblastic cells from adult rat long bones. It is unknown whether the addition of rhTGF-beta1 changes the material properties of this alpha-tricalcium-phosphate (alpha-TCP)/tetracalcium-phosphate-monoxide (TeCP)/dicalcium-phosphate-dihydrate (DCPD) cement, and what the characteristics of the release of rhTGF-beta1 from this CPC are. Therefore, in the present study we determined the release of rhTGF-beta1 from cement pellets in vitro. The possible intervening effects of the CPC modification for intermixing rhTGF-beta1 on physicochemical properties were studied by assessing the compressive strength and setting time, as well as crystallinity, calcium to phosphorus ratio, porosity and microscopic structure. Most of the previously incorporated rhTGF-beta1 in the cement pellets was released within the first 48 h. For all concentrations of rhTGF-beta1 intermixed (100 ng-2.5 mg/g CPC), approximately 0.5% of the amount of rhTGF-beta1 incorporated initially was released in the first 2 h, increasing to 1.0% after 48 h. The release of rhTGF-beta1 continued hereafter at a rate of about 0.1% up to 1 week, after which no additional release was found. The initial setting time, nor the final setting time was changed in control cement without rhTGF-beta1 (standard CPC) or in cement modified for rhTGF-beta1 (modified CPC) at 20 degrees C and 37 degrees C. Setting times were more than six times decreased at 37 degrees C compared to 20 degrees C. The compressive strength was initially low for both standard CPC and modified CPC, after which it increased between 24 h and 8 weeks. The compressive strength for the modified CPC was significantly higher compared with standard at 1, 2, and 8 weeks after mixing. X-ray diffraction revealed that both standard and modified CPC changed similarly from the original components into crystalline apatite. The calcium to phosphorus ratio as determined by an electron microprobe did not differ at all time points measured for standard CPC and modified CPC. In both standard CPC and modified CPC the separated particles became connected by crystals, forming a structure in which the particles could hardly be recognised in a densifying matrix with some small pores. The present study shows that the calcium phosphate cement is not severely changed by modification for the addition of rhTGF-beta1. The addition of rhTGF-beta1 in CPC enhances the biologic response as shown in our previous study and did not interfere with the aimed physical and chemical properties as shown in this study. We conclude that the addition of rhTGF-beta1 enlarges the potential of the CPC in bone replacement therapy.

Histological evaluation of the bone response to calcium phosphate cement implanted in cortical bone.

The aim of this study was to investigate the physicochemical and biological properties of a newly developed calcium phosphate cement (CaP cement) implanted in cortical bone. CaP cement was injected as a paste into tibia cortical bone defects in goats. Polymethylmethacrylate (PMMA) bone cement was used as a control. The animals were killed after 3 days, 2, 8, 16 and 24 weeks. X-ray diffraction and Fourier transform infrared spectroscopy performed at retrieved samples showed that the CaP cement had set as a carbonate apatite and remained stable over time. Light microscopic evaluation showed that after 2 weeks the cement was in tight contact with the bone without any inflammatory reaction or fibrous encapsulation. At later time points, the CaP cement implants were totally covered by a thin layer of bone and osteoclasts, present at the interface, which were clearly resorbing the cement. At locations where CaP cement was resorbed, new bone was deposited. Transmission electron microscopy revealed that indeed a seamless contact existed between CaP cement and bone, as characterized by the occurrence of an electron dense line of 50-60 nm thick that covered the CaP cement. Osteoblasts, in contact with the cement, were depositing new bone. Although the bulk of the material was still in situ after 24 weeks, the progressive osteoclast resorption of the cement followed by new bone formation suggests that all of the material may be replaced eventually. In contrast to the CaP cement, the PMMA reference cement was always surrounded by a thin fibrous capsule. The results indicate that the investigated CaP cement is biocompatible, osteoconductive as well as osteotransductive and is a candidate material for use as a bone substitute.