Comments on the publication "A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone".
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Bone and Bones↗
Biomedical subjects
Pierre Hardouin
Publications and source records attributed to Pierre Hardouin.
Runx2 regulates the expression of GNAS on SaOs-2 cells.
Runx2 is a key regulator of osteoblast-specific gene expression and controls the expression of multiple target genes during osteoblast differentiation. Although some transcriptional targets for Runx2 are known, it is believed that the osteogenic action of Runx2 is mediated by additional target genes, and increasing studies are performed in order to identify such Runx2-responsive genes. To identify genes following the inhibition of Runx2 in osteoblastic cell line, SaOs-2 was stably transfected with a dominant negative mutant of Runx2 (Deltacbfa1) under the control of a strong promoter. Comparison of gene expression patterns by differential display on selected SaOs-2 clones allowed us to observe that GNAS mRNA which encodes for the Gsalpha protein is overexpressed (5 to 8 fold) in cells presenting high levels of Deltacbfa1. This overexpression was also observed at the protein level and seemed to be reflected by an increased basal cAMP level. Gel shift experiments performed in this study indicate that Runx2 is able to bind to the promoter of GNAS, suggesting a direct regulation at the transcriptional level. Well-described GNAS mutations like fibrous dysplasia or Albright hereditary osteodystrophy are linked to abnormality in osteoblast function, and numerous evidences showed that Gsalpha coupled adrenergic receptors increase the expression of osteotrophic factors and regulate bone mass. Regulation of Gsalpha protein by Runx2 seems to be of particular interest considering the increasing evidences on bone metabolism regulation by G proteins.
Animals↗
Clinical, radiological and histological study of the failure of cervical interbody fusions with bone substitutes.
Few histological studies on bone substitutes in human cervical spine are available and the biological processes of bone substitutes are not well documented. The authors studied four failure cases of cervical interbody fusion: two cases with hydroxyapatite (HA), one case with beta-tricalcium phosphate ceramic (beta-TCP) and one case with xenograft (bovine bone). Clinical data showed that all the patients experienced neck pain with or without numbness of upper extremity due to fusion failure. Successful fusions were achieved after the salvage surgeries in which autograft were used. Radiographs showed that radiolucent lines were present in all cases. Two HA substitutes fractured without complications. One of them sank into the vertebral body. Some small beta-TCP fragments were found under the microscope. Histological study demonstrated only a few newly formed bones at the interface of the substitutes. The fragments of HA were encapsulated by fibrous tissue. The degradation process and bone regeneration were more active in beta-TCP than in HA. The intertrabecular spaces of bovine bone were filled with fibrous tissue. The results suggest that a porous calcium phosphate ceramic with special design might assure bone ingrowth and meet the mechanical requirements in cervical interbody fusion. The complications of these materials in the cervical spine should be highlighted.
Adult↗
Evaluation of the osteogenesis and biodegradation of porous biphasic ceramic in the human spine.
The histological reports on porous biphasic calcium phosphate ceramic (PBC) in human spine are limited. The osteogenesis and biodegradation of PBC are insufficiently known in human. In present study, the undecalcified histological study was carried out on 20 samples retrieved from posterior spinal fusion in order to reveal the osteogenesis and biodegradation of the PBC in human spine. The quantitative study was performed in 14 samples with sufficient size. Newly formed bone was found in all the samples. More new bone was formed in those samples closely in contact with autogenous bone. The PBC degradation particles were present both in the macrophages and around the tissue. However, those phenomena were highly variable among the samples. New bone formation increased with time and decreased with age. The PBC degradation decreased with age, but it did not differ greatly with time. New bone formation was higher and the residual material was lower in the fusion group than that in non-fusion group. The PBC is a kind of osteoconductive material and do not transform into new bone after a relatively long time. The PBC should be well mixed with the autogenous bone in order to achieve high new bone colonization. The PBC degradation particles and related active phagocytotic activity have been noted.
Absorbable Implants↗
Quantitative kinetic analysis of gene expression during human osteoblastic adhesion on orthopaedic materials.
Little information was found in the literature about the expression on hydroxyapatite (HA) materials of genes specific of cellular adhesion molecules although more were found on titanium-based substrates. Hence, the goal of this work was to study by a kinetic approach from 30 min to 4 days the adhesion of Saos-2 cells on microporous (mHA) and non-microporous hydroxyapatite (pHA) in comparison to polished titanium. Our strategy associated the visualization of adhesion proteins inside the cells by immunohistochemistry and the quantitative expression of genes at mRNA level by real-time PCR. The cell morphology was assessed using scanning electron microscopy and the number of cells thanks to biochemical techniques. The cellular attachment was the highest on mHA from 30 min to 24 h although the cell growth on mHA was the lowest after 4 days. Generally, the Saos-2 osteoblastic cells morphology on mHA was radically different than on other surfaces with the particularity of the cytoplasmic edge, which appeared un-distinguishable from the surface. The revelation by specific antibodies of proteins of the cytoskeleton (actin) and the focal adhesions (FAK, phosphotyrosine) confirmed that adhesion and spreading were different on the 3 materials. The actin stress fibres were less numerous and shorter on mHA ceramics. Cells had more focal contacts after 4 h on mHA compared to other substrates but less after 24 h. The highest values of total proteins were extracted from mHA at 0.5 and 24 h and from pHA at 1, 4, and 96 h. The alphav and beta1 integrin, actin, FAK, and ERK gene expression were found to be different with adhesion time and with materials. C-jun expression was comparable on mHA, titanium and plastic but was largely higher than on pHA at 0.5 and 1 h. On the contrary, c-fos expression was the highest on pHA after 0.5 h and the lowest after 1h. This difference between c-fos and c-jun expression on pHA after 0.5 h could be related to the fact that these two genes may differ in their signalling pathways. The expression of the alkaline phosphatase gene after 4 days was lower on mHA compared to other materials demonstrating that the microstructure of the mHA ceramic was not favourable to Saos-2 cells differentiation. Finally, it was demonstrated in this study that HA and titanium surfaces influence as well gene expression at early times of adhesion as the synthesis of adhesion proteins but also proliferation and differentiation phases. Indeed, the signal transduction pathways involved in adhesion of Saos-2 cells on HA and titanium were confirmed by the sequential expression of alphav and beta1 integrins, FAK, and ERK genes followed by the expression of c-jun and c-fos genes for proliferation and alkaline phosphatase gene for differentiation.
Base Sequence↗
Influence of hydroxyapatite microstructure on human bone cell response.
Microstructure of calcium phosphate ceramics has been shown to influence long-term in vitro cellular events like proliferation and differentiation, and to favor bone integration in vivo. As long-term cellular events are known to be dependent of early cell adhesion events, we decided to study the in vitro influence of the microstructure of a microporous hydroxyapatite (mHA) and a nonmicroporous hydroxyapatite (pHA) ceramic on serum protein adsorption and SaOs-2 human bone cells attachment after 30 min, 1, 4, and 24 h and cell growth after 96 h. Plastic coverslips were used as controls. Hydroxyapatite composition of mHA and pHA was confirmed by X-ray diffraction and Fourier transform infra-red spectroscopy. The surface energies of ceramics were calculated from contact-angle measurements in di-iodomethane, water or complete culture medium. The total surface energy was 44.8 mJ/m(2) for pHA and 48.7 mJ/m(2) for plastic. The contact-angle measurement was impossible on mHA likely because they displayed 12% of open microporosity, pHA ceramic exhibiting only closed pores (2.5%). Moreover, the roughness amplitude was largely higher on mHA (Sa = 4.35 microm) than on pHA (Sa = 0.065 microm) and plastic (Sa = 0.042 microm). Three different techniques were used to evaluate protein adsorption on the ceramics. SDS-PAGE of desorbed proteins demonstrated that more proteins desorbed from mHA (66.02 microg/m(2)) than from pHA (17.2 microg/m(2)) or plastic (0.08 microg/m(2)). A new method was used to evaluate in situ the quantity of adsorbed total proteins: the temperature-programmed desorption (TPD) analysis coupled with mass spectrometry. The TPD analysis confirmed that 10-fold more proteins adsorbed on mHA compared with those on pHA. A direct immunolabeling on ceramics revealed than more fibronectin and serum albumin adsorbed on microporous ceramic than on dense ceramic. The morphology of SaOs-2 cells was the same on all the substrates after 30 min. At later time points, cell morphology on mHA was radically different than on other surfaces, with the particularity of the cytoplasmic edge that appeared undistinguishable from the surface. Only the extremity of the cells and lamellipodia were visible. Cells seemed like "adsorbed" by the mHA surface, whereas on plastic and pHA surfaces the cells displayed classical aspects of polygonal spreading. The cells displayed on mHA the highest initial attachment potential after 30 min, 1, 4, 24 h but the lower proliferation potential after four days. This study confirms that a microporous ceramic surface can modulate the adsorption of proteins and further the adhesion and proliferation of human bone cells.
Adsorption↗
Study of serum factors potentially involved in the pathogenesis of heterotopic bone formation after severe brain injury.
OBJECTIVE: To look for serum factors detectable early after head injury and predictive of heterotopic bone formation. PATIENTS AND METHODS: In this prospective study of a homogeneous population of 31 men with severe brain injury, blood samples were obtained 3 months after the accident, and levels of serum factors influenced by bone metabolism were compared between patients with and without heterotopic bone formation. As extensive fractures can influence serum factors, the patients without heterotopic bone formation were divided into two groups based on whether they had major fractures. Radionuclide bone scanning was used to validate patient classification. RESULTS: The group with heterotopic bone formation had significantly higher serum alkaline phosphatase levels (P < 0.01) and significantly lower serum leptin levels (P < 0.01), as compared to the other two groups. Body mass index and serum creatinine were comparable in the three groups. CONCLUSION: Leptin may be associated with the development of heterotopic bone formation. The antiosteogenic effect of leptin mediated by hypothalamic neurons may be impaired by hypothalamic damage related to severe brain injury.
Adult↗
Biomaterial challenges and approaches to stem cell use in bone reconstructive surgery.
As life expectancy increases, so does the need to treat large bone defects. New biomaterials combined with osteogenic cells are now being developed as an alternative to autogenous bone grafts. The goal is to make the stem cells adhere to the scaffold, and then grow to differentiate into functional osteogenic cells and organize into healthy bone as the scaffold degrades. Decisive improvements have been made in the fields of stem cell biology, 3-D scaffold fabrication and tissue engineering, but the ideal bone substitute that fulfils all functional and safety requirements has yet to be developed.
Biocompatible Materials↗
Migration of polyethylene particles around nonloosened cemented femoral components from a total hip arthroplasty-an autopsy study.
Three cemented femoral components from total hip arthroplasties retrieved at autopsy were examined in regard to the migration of birefringent polyethylene particles across the fixation interfaces. They were obtained from two patients who died from unrelated causes. They had been in place, respectively, for 8 and 9 years (first patient) and for 9 years (second patient). Osteolysis was present in the acetabular side only. The femoral components were not loosened. Histologic sections were performed at four levels of the femoral components, and polyethylene particles were counted with the use of a semiautomatic image analyzer. Sections below the tip of the stem were treated by deorganification and the lysate was centrifuged in the attempt to isolate wear debris. Particles were found at both interfaces (bone-cement and metal-cement) and in the cancellous bone between the trabeculae. They were also present in the Haversian canals of the cortical bone. Particles of various sizes were found in the medullary canal beneath the tip of the stem. In scanning electron microscopy, they appeared to have irregular shapes. Wear debris can migrate across the cement-bone interface of nonloosened implants. They appear to progress through the porosity of the cancellous bone.
Aged↗
Gene expression in human osteoblastic cells from normal and heterotopic ossification.
Heterotopic ossification (HO), a possible complication of head injury, develops in sites where it is not normally present like at the vicinity of joints. It may cause pain, decrease motion and in severe cases complete joint ankylosis requiring surgical intervention. To our knowledge, no study has been made to analyze HO at the molecular level on human biopsies, whereas its etiology remains to be determined. We defined a procedure of cell fractionation from bone resections and developed quantitative RT-PCR to compare genetic expression patterns between human normal osteoblasts and heterotopic ossification forming cells. This quantitative study demonstrated a specific and strong overexpression of osteocalcin mRNA in HO-isolated cells associated with a significant upregulation of type 1 collagen and osteonectin mRNA while histological analysis showed only small cellular variations. Our results give a first molecular characterization of heterotopic ossification and we conclude that such overexpressions in HO-isolated cells could be associated with the high activity of this pathological bone.
Adult↗
Relationship between bioceramics sintering and micro-particles-induced cellular damages.
We performed experimental studies to confirm the hypothesis that cellular damages occurring around implanted biphasic bioceramics could be related to a micro-particles release because of an insufficient sintering. First, an in vitro cytotoxicity study was performed on four biphasic ceramic (BCP) samples. Without treatment of the extraction medium, a cytotoxicity was observed, although after centrifugation this cytotoxicity disappeared in all samples. Second, micro-particles of hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and 40% beta-TCP/60%HA mixture were used for a cell inhibition study. A decrease of cell viability was observed with the increase in particles concentration. At 10000 particles per cell, the viability and proliferation were completely inhibited. Third, HA, beta-TCP and BCP ceramic granules were implanted in rabbit femoral cavities for 12 weeks. No degradation of HA granules was observed. The degradation was higher for beta-TCP (40%) than for BCP (5%). On the other hand, new bone formation was significantly higher for beta-TCP (21%) and HA (18%) than for BCP (12%). More micro-particles were formed around BCP granules than around beta-TCP, and phagocytised by macrophages. The release of ceramic micro-particles could be related to the sintering process. BCP ceramic have to be sintered at only 1160 degrees C. Consequently, HA micro-particles of BCP ceramic are incompletely sintered and easily released after immersion or implantation. The microparticles could be at the origin of local inflammation and cell damage and could perhaps modify osteogenesis. Attention must be paid to this problem especially with BCP ceramics because of the sintering difficulties of this bioceramic.
Animals↗
The influence of culture conditions on extracellular matrix proteins synthesized by osteoblasts derived from rabbit bone marrow.
The influence of culture conditions on the extracellular matrix (ECM) protein expressions of rabbit bone marrow stromal cells has been studied. The focus was on the effects of two kinds of sera, fetal calf serum (FCS) and Ultroser, on cells treated with dexamethasone. The induction of osteoblastic differentiation by dexamethasone addition is confirmed, particularly when cells are cultured in FCS. Bone marrow stromal cells produce alkaline phosphatase positive CFU-F and produce ECM with some mineralized nodules. Analysis by means of two-dimensional gel electrophoresis showed important changes in the composition of ECM proteins after dexamethasone treatment. Overexpression, underexpression, and new synthesized proteins were observed. The most significant modification was linked to the synthesis of four new proteins visible in the acidic area with a low molecular weight of around 17 kDa. These proteins did not correspond to those ECM proteins known to be induced by dexamethasone. Moreover, the effect of dexamethasone on osteoblastic differentiation induction appears very limited when cells are cultured in Ultroser compared to FCS. The protein pattern with Ultroser is different to that obtained with FCS. Cells cultured in Ultroser synthesized no new protein. The different behavior of cells according to the type of medium used is discussed in terms of the osteogenic factors present in the two different sera.
Animals↗
The biodegradation mechanism of calcium phosphate biomaterials in bone.
This study was undertaken to understand the biodegradation mechanisms of calcium phosphate (Ca-P) biomaterials with different crystallization. Two types of sintered Ca-P porous ceramic (HA and beta-TCP) and a Ca-P bone cement (CPC) were implanted into cavities drilled in rabbit femoral and tibiae condyles. The results have shown that a material biodegradation was rapid in the beta-TCP and the CPC, but very weak in the HA. This biodegradation presented a decrease of material volume from the periphery to the center as well as a particle formation causing phagocytosis by numerous macrophages and multinucleated giant cells in the CPC. In the beta-TCP, there was a peripheral and central decrease of material volume as well as an absence of particle formation or visible phagocytosis. The process of biodegradation is considered to be directly influenced by the type of material crystallization. The sintered bioceramics processed at a high temperature exhibit good crystallization and are primarily degraded by a process dependent on interstitial liquids. However, the bone cement is formed by physicochemical crystallization and is degraded through a dissolution process associated with a cellular process.
Animals↗
Kyphoplasty.
A variant of vertebroplasty known as "kyphoplasty" has been suggested for correcting vertebral compression fractures. A balloon placed inside the vertebral body is inflated to create a cavity, thereby restoring vertebral body height and allowing low-pressure cement injection. This procedure is gaining popularity in the United States. Over 1000 patients had been treated by the end of 2000. However, kyphoplasty is costly (chiefly because the balloon is disposable) and has not been evaluated in carefully designed studies. Although retrospective findings have been reported as highly promising, they are not sufficient to validate this procedure. The principle is innovative and the procedure deserves further investigation as a potentially effective means of correcting loss of vertebral height. Furthermore, use of a bone substitute instead of cement deserves investigation.
Humans↗
In vitro control of human bone marrow stromal cells for bone tissue engineering.
For the clinical application of cultured human mesenchymal stem cells (MSCs), cells must have minimal contact with fetal calf serum (FCS) because it might be a potential vector for contamination by adventitious agents. The use of human plasma and serum for clinical applications also continues to give rise to considerable concerns with respect to the transmission of known and unknown human infectious agents. With the objective of clinical applications of cultured human MSCs, we tested the ability of autologous plasma, AB human serum, FCS, and artificial serum substitutes containing animal-derived proteins (Ultroser G) or vegetable-derived proteins (Prolifix S6) to permit their growth and differentiation in vitro. To conserve as much autologous plasma as possible, we attempted to mix it at decreasing concentrations with the serum substitute containing vegetable-derived mitogenic factors. Under control conditions, by day 10 all the fibroblast colony-forming units (CFU-Fs) were alkaline phosphatase (ALP) positive. However, their number and size were highly variable among donors. Better CFU-F formation was obtained with Ultroser G, and with human AB serum and autologous plasma mixed at, respectively, 5 and 1% with Prolifix S6. The effects of these mixtures on CFU-F formation demonstrate synergy, with the human serum or plasma supplying the factors that favor differentiation of MSCs while Prolifix S6 supplies the mitogenic factors. Finally, we demonstrated the possibility of controlling human MSC growth and differentiation in vitro. Notably, by means of a minimal quantity of human serum or human plasma mixed with a new serum substitute containing vegetable-derived proteins, we displayed growth and differentiation of human MSCs comparable to that obtained with FCS or serum substitutes containing animal-derived proteins. These results will have crucial significance for future applications of cultured human MSCs in bone tissue engineering.
Alkaline Phosphatase↗
New Injectable Composites for Bone Replacementd.
A new class of biomaterials is emerging: injectable composites for bone replacement. They include injectable ceramics and injectable calcium phosphate hydraulic cements, such as calcium deficient hydroxyapatite cements, dahllite cements, or brushite cements. Their main advantages are their biocompatibility, resorbability, osteoconductivity, and injectability, which allows a delivery with a syringe and a needle through a percutaneous approach. Furthermore, they can be used for the controlled delivery of antibiotics or bone morphogenetic proteins. Their mechanical properties remain nonsatisfactory as compared to orthopedic cement [polymethylmetacrylate (PMMA)], but these materials still have a great potential for improvement. There is a large field of potential clinical applications for these composites, especially in implantology, bone surgery, traumatology, interventional radiology, and rheumatology. However, the biological properties of a limited number of these compounds have been studied so far and further biological evaluations, as well as rigorous clinical studies, remain to be made.
Journal Article↗