Interleukin-6 expression by osteoblast-like MG63 cells challenged with four acrylic bone cements

Periprosthetic osteolysis is a major clinical problem in total hip and total knee arthroplasty and polymethylmethacrylate (PMMA) is a possible etiologic factor. Recently, increasing importance was ascribed to interleukin-6 (IL-6) as an agent favouring bone resorption. The aim of the present study was to investigate the role of bone cements on IL-6 production by MG63. The effect of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2, and CMW 3) on the protein release and mRNA expression of IL-6 in osteoblast-like cell line MG63 was examined using IL-1beta (0.2 microg ml(-1)) as the positive control. The extracts in minimum essential medium (MEM) of the cements were tested, following 1-h and 7-day curing. CMW 1 and CMW 2 significantly increased the IL-6 release into the culture media (p < 0.01). The cells incubated with Sulfix-60 and CMW 3 produced no significantly different levels of IL-6 than the basal production. A positive correlation was found between the concentration of IL-6 and the contents of benzoylperoxide (p = 0.0003) and barium sulphate (p < 0.0001). MG63 expressed IL-6 mRNA constitutively, as demonstrated by the positivity of the negative controls too. We conclude that CMW 1 and CMW 2 increase the production of IL-6 in MG63 cells. The response to Sulfix-60 and CMW 3 is not significantly greater than the negative control.     

Effect of four acrylic bone cements on transforming growth factor-beta1 expression by osteoblast-like cells MG63.

Based on the hypothesis that bone cements cause changes in the production of transforming growth factor-beta 1 (TGF-beta1) by bone cells, the effects of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2 and CMW 3) were examined using the osteoblast-like cell line MG63. The extracts in MEM of the cements were tested, following 1 h- and 7 day-curing. MG63 cells seldom expressed mRNA specific for TGF-beta1 in basal conditions. The cultures expressed mRNA constantly after incubation with the extract of CMW 1 cured for 1 h. TGF-beta1 specific mRNA was seldom expressed after incubation with the other cement extracts. The release of TGF-beta1 into the conditioned medium was increased significantly by CMW 1 extract at 1 h-curing, but was not changed significantly by CMW 1 extract at 7 day-curing and by the extracts of the other cements, at both curing times. The stimulating effect of CMW 1 on the secretion of TGF-beta1, even with all the restrictions of an in vitro study of continuous cell lines, if confirmed in vivo, might favor the development of the synovial-like membrane around the implant, and therefore impair the chance of success of the prosthesis.     

Gene expression of bone-associated cytokines in MG63 osteoblast-like cells incubated with acrylic bone cement extracts in minimum essential medium

This study examined the effects of in vitro challenge with four polymerized acrylic bone cements (Sulfix-60, CMW 1, CMW 2, and CMW 3) on the expression of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-beta1) mRNAs in the osteoblast-like cell line MG63. The extracts of the cements in minimal essential medium (MEM) were tested following 1-h and 7-day curing. A semi-quantitative analysis of the cytokine-specific mRNAs was carried out by agarose gel densitometry and expression was compared with the GAPDH housekeeping gene. The ratio between cytokine gene expression and GAPDH expression was calculated. The mRNA specific for the bone-resorbing cytokines IL-1beta and IL-6 was low in basal conditions. IL-1beta mRNA increased only after incubation with the extract of CMW 1 following 1-h curing. The mRNA specific for the bone-resorbing cytokine IL-6 also increased after contact with CMW 1 at both curing times. Sulfix-60 and CMW 3 following 7-day curing, but not after 1 h, induced higher levels of IL-6 mRNA than the control. CMW 2 after 1-h curing constantly determined the expression of IL-6 mRNA, but at low levels. The mRNA specific for TGF-beta1 was also expressed by the MG63 osteoblast-like cells in basal conditions. The levels increased after contact with Sulfix-60 after 7-day curing and with CMW 1 after 1-h curing. CMW 2 after 7-day curing decreased TGF-beta1 mRNA. In conclusion, the highest expression of the cytokines IL-1beta, IL-6, and TGF-beta1 mRNA was determined by CMW 1. If the results are confirmed in vivo, the increased expression of the osteolytic cytokines induced by the bone cement might result in loosening of the prosthesis, even with all the restrictions of an in vitro study on continuous cell lines.     

Interleukin-6 expression by osteoblast-like MG63 cells challenged with four acrylic bone cements

Periprosthetic osteolysis is a major clinical problem in total hip and total knee arthroplasty and polymethylmethacrylate (PMMA) is a possible etiologic factor. Recently, increasing importance was ascribed to interleukin-6 (IL-6) as an agent favouring bone resorption. The aim of the present study was to investigate the role of bone cements on IL-6 production by MG63. The effect of four acrylic bone cements (Sulfix-60^®, CMW 1^®, CMW 2^®, and CMW 3^®) on the protein release and mRNA expression of IL-6 in osteoblast-like cell line MG63 was examined using IL-1 β (0.2 μg ml-1) as the positive control. The extracts in minimum essential medium (MEM) of the cements were tested, following 1-h and 7-day curing. CMW 1^® and CMW 2^® significantly increased the IL-6 release into the culture media (p < 0.01). The cells incubated with Sulfix-60® and CMW 3® produced no significantly different levels of IL-6 than the basal production. A positive correlation was found between the concentration of IL-6 and the contents of benzoylperoxide (p = 0.0003) and barium sulphate (p < 0.0001). MG63 expressed IL-6 mRNA constitutively, as demonstrated by the positivity of the negative controls too. We conclude that CMW 1^® and CMW 2^® increase the production of IL-6 in MG63 cells. The response to Sulfix-60^® and CMW 3^® is not significantly greater than the negative control.     

Gene expression of bone-associated cytokines in MG63 osteoblast-like cells incubated with acrylic bone cement extracts in minimum essential medium

This study examined the effects of in vitro challenge with four polymerized acrylic bone cements (Sulfix-60®, CMW 1®, CMW 2®, and CMW 3®) on the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and transforming growth factor-β1 (TGF-β1) mRNAs in the osteoblastlike cell line MG63. The extracts of the cements in minimal essential medium (MEM) were tested following 1-h and 7-day curing. A semi-quantitative analysis of the cytokine-specific mRNAs was carried out by agarose gel densitometry and expression was compared with the GAPDH house-keeping gene. The ratio between cytokine gene expression and GAPDH expression was calculated. The mRNA specific for the bone-resorbing cytokines IL-1β and IL-6 was low in basal conditions. IL-1β mRNA increased only after incubation with the extract of CMW 1® following 1-h curing. The mRNA specific for the bone-resorbing cytokine IL-6 also increased after contact with CMW 1® at both curing times. Sulfix-60® and CMW 3® following 7-day curing, but not after 1 h, induced higher levels of IL-6 mRNA than the control. CMW 2® after 1-h curing constantly determined the expression of IL-6 mRNA, but at low levels. The mRNA specific for TGF-β1 was also expressed by the MG63 osteoblast-like cells in basal conditions. The levels increased after contact with Sulfix-60® after 7-day curing and with CMW 1® after 1-h curing. CMW 2® after 7-day curing decreased TGF-β1 mRNA. In conclusion, the highest expression of the cytokines IL-1β, IL-6, and TGF-β1 mRNA was determined by CMW 1®. If the results are confirmed in vivo, the increased expression of the osteolytic cytokines induced by the bone cement might result in loosening of the prosthesis, even with all the restrictions of an in vitro study on continuous cell lines.     

Surface roughness, porosity and wettability of gentamicin-loaded bone cements and their antibiotic release

In this study, the release of gentamicin as a function of time was measured for six different gentamicin-loaded bone cements and related with the surface roughness, porosity and wettability of the cements. Initial release rates varied little between the six bone cements (CMW1, CMW3, CMW Endurance, CMW 2000, Palacos, and Palamed) and ranged from 8.6 to 14.1 microg/cm2/h. The total amounts of gentamicin released after 1 week varied between 4.0 and 5.3% of the total amount of antibiotic incorporated for the CMW cements and was 8.4% for Palacos. Palamed released after 1 week significantly more of the gentamicin incorporated (17.0%). The wettability of all cements was similar (water contact angles between 70 and 80 degrees), but the surface roughness and the porosity of the cements varied markedly. Initial release rates increased with surface roughness, although the correlation coefficient was low (0.64), while total amounts released increased linearly (correlation coefficient 0.97) with the bulk porosity of the cements. Consequently, it can be concluded that the release kinetics of gentamicin from bone cements is controlled by a combination of surface roughness and porosity.     

Staphylococcus aureus biofilm formation on different gentamicin-loaded polymethylmethacrylate bone cements

In this in vitro study, the formation of a Staphylococcus aureus biofilm on six gentamicin-loaded bone cements (CMW1, CMW3, CMW Endurance, CMW2000, Palacos, and Palamed) was determined in a modified Robbins device over a 3 days time span and related with previously (Van de Belt et al., Biomaterials 21 (2000) 1981) measured kinetics of antibiotic release by these cement brands. The influence of gentamicin release on biofilm formation was quantified by expressing the number of colony-forming units on gentamicin-loaded cement relative to the number of viable organisms on unloaded cement of the same brand. Biofilms formed on all gentamicin-loaded cements, despite the release of antibiotics, followed a consistent pattern in time with a maximum number of colony-forming units per unit cement area found between 24 and 30 h after inoculation. None of the gentamicin-loaded cements showed a reduction in biofilm formation relative to unloaded cements within 6 h after inoculation, whereas only gentamicin-loaded CMW1 and Palacos reduced biofilm formation 24 h after inoculation. Alternatively, CMW Endurance, CMW2000, and Palamed did not exhibit any initial reductions in biofilm formation, but effects started after 72, 48, and 72 h, respectively. Biofilm reduction by gentamicin-loaded CMW3 lasted the longest from 24 to 72 h. Interestingly, each cement seemed to have a different "window-of-effectiveness" with regard to reduction in biofilm formation that did not relate with the gentamicin-release kinetics. Summarising, this study demonstrates that although gentamicin loading of bone cements yields reductions in biofilm formation, S. aureus is able to grow on gentamicin-loaded bone cements.     

Production of TNF-alpha and bone resorbing activity by macrophages in response to different types of bone cement particles

We have compared the capacity of clinically relevant wear debris from seven different cement types to activate macrophages to produce TNF-alpha, IL-1beta, IL-6 and bone resorbing activity in vitro. The bone cements were: CMW 1 original (PMMA only); CMW 1RO (1 microm BaSO4; 9.2%); CMW copolymer bone cement 1 (10 microm BaSO4; 10%); CMW copolymer bone cement 2 (1 microm BaSO4; 10%); Palacos R (10 microm ZrO2; 15.6%); CMW Calcium phosphate cement 20% (10 microm tri-calcium phosphate; 20%) and CMW calcium phosphate cement 30% (10 microm tri-calcium phosphate; 30%). Cement debris was produced aseptically using a simple configuration wear test. The majority of particles were in the size range 0.1-0.5 microm for each cement type. The cement particles were co-cultured with the U937 macrophage cell line at ratios of 10 and 100 microm3 particle volumes to macrophage cell numbers for 24 h. At the 10:1 ratio the particles had no effect on the cells. At the 100:1 ratio, the major cytokine produced was TNF-alpha and there were no statistical differences between the different types of cement debris. The bone resorption activity of the co-culture supernatants was significantly greater than the control (U937 cells without particles) for particles of CMW 1RO, CMW copolymer bone cement 1, CMW copolymer bone cement 2 and Palacos R (P < 0.05, ANOVA). However there were no statistical differences between the levels of bone resoprtion evoked by these four cement types. The CMW1 original and CMW calcium phosphate containing cements failed to induce the macrophages to elaborate bone resorption activity at the 100:1 ratio. These data suggest that the addition of radio-opaque additives to bone cement may increase the capacity of the debris to induce osteolysis.     

Platelet release of transforming growth factor-beta and beta-thromboglobulin after in vitro contact with acrylic bone cements

Three methacrylate-based bone cements used for the fixation of joint prostheses were evaluated: Sulfix-60 (Sulzer Orthopedic Inc., Baar, Switzerland). CMW1 (DePuy International Ltd., England). and CMW2 (DePuy International Ltd., England). The cements after polymerization were put in contact in vitro with platelet-rich plasma. Plasma, in contact only with siliconized glass, was used as a negative control. After contact, platelet number. beta-thromboglobulin (beta-TG), and transforming growth factor-beta1 (TGF-beta1) were determined. The Student's paired t test showed that the ccments induced no significant modifications of platelet number. CMWI and Sulfix-60 determined a significant increase in beta-TG compared with the negative control. All cements determined a significant increase in TGF-beta1. Significant differences were also seen in the levels of beta-TG and TGF-beta1 between cements with a content of benzoyl peroxide < 1 (Sulfix-60) and those with a content > 1 (CMW1 and CMW2). The cement with zirconium dioxide (Sulfix-60) produced higher levels of beta-TG and TGF-beta1, compared to those with barium sulphate (CMW1 and CMW2). In conclusion, all the cements induced the secretion of TGF-beta1 CMW1 and Sulfix-60 determined also a significant release of beta-TG. Platelet activation induced by the cements from one side could contribute to the pathogenesis of deep venous thrombosis, that often occurs after prosthetic implant and is caused also by other factors, including surgical trauma and venous stasis. From the other side, activated platelets can release growth factors favoring bone formation.     

Serum from patients with ankylosing spondylitis can increase PPARD,fra-1, MMP7, OPG and RANKL expression in MG63 cells

OBJECTIVES:To explore the effects of serum from patients with ankylosing spondylitis on the canonical Wnt/β-catenin pathway and to assess whether the serum has an osteogenic effect in MG63 cells.METHODS:MG63 cells were cultured with serum from 45 ankylosing spondylitis patients, 30 healthy controls, or 45 rheumatoid arthritis patients. The relative PPARD, fra-1, MMP7, OPG and RANKL mRNA levels were measured using quantitative real-time polymerase chain reaction. Associations between gene expression and patient demographics and clinical assessments were then analyzed.RESULTS:MG63 cells treated with serum from ankylosing spondylitis patients had higher PPARD, fra-1, MMP7 and OPG gene expression than did cells treated with serum from controls or rheumatoid arthritis patients (all p<0.05). RANKL expression was higher in MG63 cells treated with serum from patients with ankylosing spondylitis or rheumatoid arthritis than in those treated with serum from controls (both p<0.05). The OPG/RANKL ratio was also higher in MG63 cells treated with serum from ankylosing spondylitis patients than in those treated with serum from controls (p<0.05). No associations were found between the expression of the five genes and the patient demographics and clinical assessments (all p>0.05).CONCLUSIONS: Serum from ankylosing spondylitis patients increases PPARD, fra-1, MMP7, OPG and RANKL expression and the OPG/RANKL ratio in MG63 cells; these effects may be due to the stimulatory effect of the serum on the Wnt pathway.     

Effect of CMW 1 bone cement on transforming growth factor-beta 1 expression by endothelial cells.

The present study examined the effects of in vitro challenge with an acrylic bone cement CMW 1 on the expression of transforming growth factor-beta 1 (TGF-beta 1) in human umbilical vein endothelial cells (HUVEC). The extracts in cell culture medium of the cements were tested, after 1 h and 7-day curing. Some cultures were also stimulated with interleukin-1 beta (IL-1 beta) or all-trans retinoic acid (ATRA). The expression of mRNA was evaluated by RT-PCR with specific primers. The release of TGF-beta 1 into the conditioned medium was evaluated by enzyme immunoassay. TGF-beta 1 mRNA was constitutively expressed by endothelial cells in the culture medium after 24 h. The incubation with the extracts of CMW 1, cured both for 1 h and 7 days, induced changes neither in mRNA expression, nor in the release of TGF-beta 1 into the conditioned medium, compared to the unstimulated cells. Even stimulation with ATRA, alone or added to the extracts at both curing times, affected neither mRNA expression nor TGF-beta 1 release, compared to the cells incubated with the cement alone or with the unstimulated cultures. The mRNA expression and the release were not changed by the stimulation with IL-1beta alone or added to the extract cured for 1 h. A significant decrease compared to the unstimulated cells was observed after the addition of IL-1 beta to the extract cured for 7 days. It was concluded that CMW 1 extract did not significantly modify TGF-beta 1 expression after 1-h curing, or after 7-day curing. Incubation with CMW 1 added with ATRA did not produce any changes in TGF-beta 1 synthesis. Incubation with cement extract after 7-day curing added with IL-beta 1 produced a significant reduction in TGF-beta 1 release.     

Evaluation of the effect of seven acrylic bone cements on erythrocytes and plasmatic phase of coagulation

The haemolytic activity and the effect on the plasmatic phase of coagulation of seven bone cements were evaluated (Sulfix-60 from Sulzer Orthopedic Inc., a bone cement at low viscosity from Zimmer, a bone cement dough-type from Zimmer, Palacos R from Merck, CMW1, CMW2 and CMW3 from DePuy International Ltd.). Haemolytic activity was tested by adding the cement extracts in phosphate buffered saline to a suspension of erythrocytes. After 4 h incubation at 37 degrees C, the haemoglobin concentration was determined on the supernatants by colorimetric method. The effect on the plasmatic phase of coagulation was tested by adding the cement extracts in saline to human plasma. After 30 min incubation at room temperature activated partial thromboplastin time (APTT) was determined. All extracts induced non-significant variations of haemoglobin concentration and APTT. It was concluded that the tested cement extracts do not induce haemolysis and do not activate the intrinsic pathway of coagulation, at least in the tests that were performed.     

Effects of bone cement extracts on the cell-mediated immune response.

The aim of the study was to evaluate some aspects of the immunocompatibility of 10 acrylic bone cements. Mononuclear cells harvested from healthy individuals were cultured with cement extracts which were tested to assess their effect on the viability of lymphocytes, unstimulated and phytohaemoagglutinin (PHA)-stimulated, activating resting lymphocytes, and changing the reactivity of PHA-stimulated lymphocytes. After 24 h the extracts did not increase the percentage of dead cells in unstimulated or PHA-stimulated lymphocytes. The early apoptotic events of culture were evaluated after 4 and 24 h in PHA-stimulated lymphocytes: at 4 h three cements, namely Zimmer-dough type, Palacos R and CMW-1, increased significantly the percentage of apoptotic cells, while at 24 h no differences were found. Cement extracts did not activate the resting lymphocytes, whereas the response of the PHA-stimulated cells was significantly modified. All cements decreased the expression of the interleukin 2 receptor (CD25) and the lymphocyte proliferation, whereas only two materials (Zimmer-dough type, CMW 1) affected the expression of early activation antigen (CD69). These findings show that the products released from bone cement are not able, by themselves, to elicit a specific immune response; on the contrary they hamper the function of lymphocytes activated by an exogenous stimulus.     

PMMA bone cement containing a quaternary amine comonomer with potential antibacterial properties

An iodinated quaternary amine dimethacrylate monomer was synthesized and incorporated as a comonomer in acrylic bone cements. Bone cement is used in orthopaedic surgery and imparting antibacterial properties to the cement can be beneficial in the lowering of bacterial infection post surgery. PMMA based bone cements were modified by copolymerising the monomer methylmethacrylate (MMA) with a quaternary amine dimethacrylate by using the redox initiator activator system as used for curing commercial bone cements. The cements were prepared using the commercial PMMA bone cement CMW and the liquid component was modified with the amine to render antimicrobial properties to the cement. The physical, mechanical, and antimicrobial properties of the modified cements were evaluated; in addition, the viability of the cement to function as a orthopaedic cement was also established, especially with an advantage of it being radiopaque, due to the inclusion of the iodine containing quaternary amine. The cytotoxicity of the modified cements were tested using a human cell model and the results indicated that the cells remained metabolically active and proliferated when placed in direct contact with the experimental cement specimens. The cements and their eluants did not evoke any cytotoxic response.     

The release of gentamicin from acrylic bone cements in a simulated prosthesis-related interfacial gap

Gentamicin is added to polymethylmethacrylate bone cements in orthopedics as a measure against infection in total joint arthroplasties. Numerous studies have been published on gentamicin release from bone cements, but none have been able to estimate the local concentrations in the prosthesis-related interfacial gap, present after implantation. The aim of this study was to develop a method allowing determination of antibiotic release in such a gap. Two-hundred-micrometer-wide gaps with a volume of 6 microl and a surface area of 0.6 cm2 were created by inserting stainless-steel strips in gentamicin-loaded bone cement plugs prior to polymerization. After hardening, the gap surface was exposed to 6 microl or 10 ml of phosphate-buffered saline. Within 2 h, gentamicin concentrations in the gaps reached around 4000 microg/ml for 4 different CMW and Palamed cements and 2500 microg/ml for Palacos R. Concentrations measured in the larger volume were several hundred times lower than in the gaps. This simulated prosthesis-related interfacial gap model offers new insights in the clinical efficacy of antibiotic-loaded bone cements. It is demonstrated that concentrations up to 1000-fold the antibiotic resistance levels for most bacterial strains causing implant infection can be achieved in a realistic in vitro model.     

Biocompatibility and other properties of acrylic bone cements prepared with antiseptic activators

Acrylic bone cements prepared with activators of reduced toxicity have been formulated with the aim of improving the biocompatibility of the final material. The activators used were N,N-dimethylaminobenzyl alcohol (DMOH) and 4,4'-dimethylamino benzydrol (BZN). The toxicity, cytotoxicity, and antiseptic action of these activators were first studied. DMOH and BZN presented LD50 values 3-4 times higher than DMT, were less cytotoxic against polymorphonuclear leucocytes, and possessed an antimicrobial character, with a high activity against the most representative microorganisms involved in postoperative infections. The properties of the acrylic bone cements formulated with DMOH and BZN were evaluated to determine the influence of these activators on the curing process and the physicochemical characteristics of the cements. A decrease of the peak temperature was observed for the curing with DMOH or BZN with respect to that of one commercially available formulation (CMW 3). However, residual monomer content and mechanical properties in tension and compression were comparable to those of CMW 3. The biocompatibility of acrylic bone cements containing DMOH or BZN was studied and compared with CMW 3. To that end, intramuscular and intraosseous implantation procedures were carried out and the results were obtained from the histological analysis of the surrounding tissues at different periods of time. Implantation of rods of cement into the dorsal muscle of rats showed the presence of a membrane of connective tissue, which increased in collagen fibers with time of implantation, for all formulations. The intraosseous implantation of the cements in the dough state in the femur of rabbits, revealed a higher and early osseous neoformation, with the presence of osteoid material surrounding the rest of the cured material, for the cement prepared with the activator BZN in comparison with that obtained following the implantation of the cement cured with DMOH or DMT (CMW 3).     

Platelet activation after in vitro contact with seven acrylic bone cements

Seven acrylic bone cements were evaluated: Cemex Rx (Tecres S.p.a., Italy), Cemex Isoplastic (Tecres S.p.a., Italy), Zimmer Low Viscosity Cement (L.V.C. , Zimmer, IN, USA), Zimmer bone cement—dough type (Zimmer, IN, USA), CMW 3 (DePuy International Ltd., UK), Cerim LT (Cremascoli S.r.l., Italy), and Palacos R (Merck, Wehreim, Germany). The cements after polymerization were put in contact in vitro with platelet-rich plasma. Plasma in contact only with siliconated glass was used as the negative control. After contact, platelet number, β-thromboglobulin (β-TG), and transforming growth factor-β1 (TGF-β1) were determined. The Wilcoxon signed rank test showed Palacos R and L.V.C. induced a significant decrease of platelet number compared with the negative control. All cements determined a significant increase in β-TG. CMW 3 , Palacos , L.V.C. , and Zimmer dough type determined a significant increase in TGF-β1 compared with the negative control.     

Platelet activation after in vitro contact with seven acrylic bone cements

Seven acrylic bone cements were evaluated: Cemex Rx (Tecres S.p.a., Italy), Cemex Isoplastic (Tecres S.p.a., Italy), Zimmer Low Viscosity Cement (L.V.C., Zimmer, IN, USA), Zimmer bone cement - dough type (Zimmer, IN, USA), CMW (DePuy International Ltd., UK), Cerim LT (Cremascoli S.r.l., Italy), and Palacos (Merck, Wehreim, Germany). The cements after polymerization were put in contact in vitro with platelet-rich plasma. Plasma in contact only with siliconated glass was used as the negative control. After contact, platelet number, beta-thromboglobulin (beta-TG), and transforming growth factor-beta1 (TGF-beta1) were determined. The Wilcoxon signed rank test showed Palacos R and L.V.C. induced a significant decrease of platelet number compared with the negative control. All cements determined a significant increase in beta-TG. CMW 3, Palacos, L.V.C., and Zimmer dough type determined a significant increase in TGF-beta1 compared with the negative control.     

Effect of CMW 1 bone cement on transforming growth factor-beta 1 expression by endothelial cells

The present study examined the effects of in vitro challenge with an acrylic bone cement CMW1 on the expression of transforming growth factor-beta 1 (TGF-β1) in human umbilical vein endothelial cells (HUVEC). The extracts in cell culture medium of the cements were tested, after 1 h and 7-day curing. Some cultures were also stimulated with interleukin-1β (IL-1β) or all-trans retinoic acid (ATRA). The expression of mRNA was evaluated by RT-PCR with specific primers. The release of TGF-β1 into the conditioned medium was evaluated by enzyme immunoassay. TGF-β1 mRNA was constitutively expressed by endothelial cells in the culture medium after 24 h. The incubation with the extracts of CMW 1, cured both for 1 h and 7 days, induced changes neither in mRNA expression, nor in the release of TGF-β1 into the conditioned medium, compared to the unstimulated cells. Even stimulation with ATRA, alone or added to the extracts at both curing times, affected neither mRNA expression nor TGF-β1 release, compared to the cells incubated with the cement alone or with the unstimulated cultures. The mRNA expression and the release were not changed by the stimulation with IL-1β alone or added to the extract cured for 1 h. A significant decrease compared to the unstimulated cells was observed after the addition of IL-1β to the extract cured for 7 days. It was concluded that CMW 1 extract did not significantly modify TGF-β1 expression after 1-h curing, or after 7-day curing. Incubation with CMW1 added with ATRA did not produce any changes in TGF-β1 synthesis. Incubation with cement extract after 7-day curing added with IL-β1 produced a significant reduction in TGF-β1 release.     

Setting properties and in vitro bioactivity of strontium-enriched gelatin-calcium phosphate bone cements

Strontium is known to reduce bone resorption and stimulate bone formation. We have investigated the effect of strontium on the setting properties and in vitro bioactivity of a biomimetic gelatin-calcium phosphate bone cement. Gelatin-alpha-TCP powders, with a gelatin content of 15 wt %, were prepared by grinding and sieving the solid compounds obtained by casting gelatin aqueous solutions containing alpha-TCP. 5 wt % of CaHPO(4).2H(2)O were added to the cement powders before mixing with the liquid phase, with a L/P ratio of 0.3 mL/g. Strontium was added as SrCl(2).6H(2)O in different amounts up to 5 atom %. X-ray diffraction analysis, mechanical tests, and SEM investigations were carried out on the cements after different times of soaking in physiological solution. The presence of strontium affects both the initial and the final setting times of the cements, which increase with the ion content. The microstructural modifications observed in the SEM micrographs of the fractured surfaces are in agreement with the increase of the total porosity, and with the slight reduction of the compressive strength of the aged cements, on increasing strontium content. The rate of transformation of alpha-TCP into calcium deficient hydroxyapatite increases on increasing strontium content. SEM reveals that MG63 osteoblasts grown on the cements show a normal morphology and biological tests demonstrate very good rate of proliferation and viability in every experimental time. In particular, strontium stimulates Alkaline Phosphatase activity, Collagen type I, osteocalcin, and osteoprotegerin expression.