Biomedical subjects

H Zreiqat

Publications and source records attributed to H Zreiqat.

At least 19 recordsLinked to original sources

PubMed2005

Bioceramics composition modulate resorption of human osteoclasts.

Biomaterials used in bone regeneration are designed to be gradually resorbed by the osteoclast and replaced by new bone formed through osteoblastic activity. The aim of the present study is to analyze the role of osteoclasts in the resorption process. The attachment of human osteoclasts and the appearance of their resorption lacunae, when cultured on either the resorbable crystalline, calcium orthophosphate materials or on the long-term stable bioceramic material was investigated. The resorbable materials contain Ca10[K,Na](PO4)7 (AW-Si) and Ca2KNa(PO4)2 (GB14, GB9 & D9/25) as their main crystal phases, however they differ in their total solubility. These differences result from small variations in the composition. The long-term stable material consist of about 30% fluorapatite beside calcium zirconium phosphate (Ca5(PO4)3F + CaZr4(PO4)6) and shows a very small solubility. AW-Si has an alkali containing crystalline phase, Ca10[K,Na](PO4). While GB14, GB9 and D9/25 contain the crystalline phase Ca2KNa(PO4)2 with small additions of crystalline and amorphous diphosphates and/or magnesium potassium phosphate (GB14). D9/25 and AW-Si is less soluble compared to GB14, and GB9 among the resorbable materials. Resorbable and long-term stable materials vary in their chemical compositions, solubility, and surface morphology. Osteoclasts modified the surface in their attempts to resorb the materials irrespective of the differences in their physical and chemical properties. The depth and morphology of the resorption imprints were different depending on the type of material. These changes in the surface structure created by osteoclasts are likely to affect the way osteoblasts interact with the materials and how bone is subsequently formed.

Biocompatible Materials↗

PubMed2005

The effect of surface chemistry modification of titanium alloy on signalling pathways in human osteoblasts.

Establishing and maintaining mature bone at the bone-device interface is critical to the long-term success of prosthesis. Poor cell adhesion to orthopaedic and dental implants results in implant failure. Considerable effort has been devoted to alter the surface characteristics of these biomaterials in order to improve the initial interlocking of the device and skeleton. We investigated the effect of surface chemistry modification of titanium alloy (Ti-6Al-4V) with zinc, magnesium or alkoxide-derived hydroxy carbonate apatite (CHAP) on the regulation of key intracellular signalling proteins in human bone-derived cells (HBDC) cultured on these modified Ti-6Al-4V surfaces. Western blotting demonstrated that modifying Ti-6Al-4V with CHAP or Mg results in modulation of key intracellular signalling proteins. We showed an enhanced activation of Shc, a common point of integration between integrins and the Ras/Mapkinase pathway. Mapkinase pathway was also upregulated, suggesting its role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the Mg and CHAP modified Ti-6Al-4V. Thus surface modification with CHAP or Mg may contribute to successful osteoblast function and differentiation at the skeletal tissue-device interface.

Alloys↗

PubMed2004

Effect of rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins in vitro.

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation because it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins by human bone-derived cells (HBDCs) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, two materials with a crystalline phase Ca(2)KNa(PO(4))(2) and with a small amorphous portion containing either magnesium potassium phosphate (material denominated GB14) or silica phosphate (material denominated GB9), and a calcium phosphate bone cement (material denominated Biocement D). HBDCs were grown on the substrata for 3, 7, 14, and 21 days, counted, and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and bone sialoprotein). All substrates supported continuous cellular growth for 21 days. In the presence of GB14 and Biocement D specimens cell proliferation was reduced and cell differentiation increased. At day 21, the greatest number of cells was found on GB9 expressing significantly higher levels of bone-related proteins than cells grown on all other surfaces. Because all novel materials facilitated the expression of the osteoblastic phenotype at least as much as TCP and the polystyrene control, these biomaterials can be regarded as excellent candidate bone substitute materials. GB9 induced the highest proliferation and cellular differentiation after 21 days of incubation, suggesting that this material may possess a higher potency for enhancing osteogenesis than TCP.

Adolescent↗

PubMed2004

Regulation of osteoclast activity in peri-implant tissues.

Implants, particularly joint replacement prostheses, are one of the great success stories of modern medicine. However, too many implants fail prematurely, mainly due to aseptic bone loss around the implant. This paper reviews our current understanding of the role of osteoclasts in this peri-implant bone lysis. Prosthetic particles, often produced by articulating prostheses, are one of the major causes of elevated osteoclast lysis of peri-implant bone. Over the past decade there have been major advances in our understanding of the factors that regulate osteoclast activity, many of which were found to be important in osteoclast formation and activity in the peri-implant tissues. These factors are targets of a number of recently developed drugs that have been used successfully to prevent and treat peri-implant bone lysis in experimental models. Treatments such as these are being used in a number of bone loss pathologies in humans and have the potential for successful treatment of peri-implant osteolysis. In addition, understanding how different biomaterials influence the expression of key osteoclastogenic factors may allow us to select biomaterials for implantation that will last the lifetime of the recipient.

Animals↗

PubMed2004

The modulation of osteogenesis in vitro by calcium titanium phosphate coatings.

Calcium phosphate coated titanium and titanium alloy are widely used as dental and orthopaedic implants. This study examines the effect of novel calcium titanium and calcium titanium zirconium phosphates suitable for plasma-spraying onto titanium substrata on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that on native titanium and hydroxyapatite-coated titanium. Test materials were an acid etched and sand-blasted titanium surface (Ti-DPS), a plasma-sprayed hydroxyapatite coating (HA), and five materials which were created from CaTi(4)(PO(4))(6) (CTP) and CaZr(4)(PO(4))(6) (CZP): sintered CaTi(4)(PO(4))(6) (CTP-S1), sintered 46CaO.23TiO(2).31P(2)O(5) (CTP-S2), sintered CaTiZr(3)(PO(4))(6), (CTZP-S1), sintered 46CaO.23ZrO(2).31P(2)O(5) (CTZP-S2) and sintered 55CaO.20TiO(2).31P(2)O(5) (CTP-S3). HBDC were grown on the substrata for 3, 7, 14 and 21 d, counted and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrates significantly affected cellular growth and the temporal expression of an array of bone-related genes and proteins. At 14 and 21 d, cells on CTP-S3 displayed significantly enhanced expression of all osteogenic mRNAs. Surfaces of CTP-S1 and CTP-S3 had the most effect on osteoblastic differentiation inducing a greater expression of an array of osteogenic markers than recorded for cells grown on Ti-DPS and HA, suggesting that these novel materials may possess a higher potency to enhance osteogenesis.

Alkaline Phosphatase↗

PubMed2004

The functional expression of human bone-derived cells grown on rapidly resorbable calcium phosphate ceramics.

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.

Bone Substitutes↗

PubMed2004

Factors regulating osteoclast formation in human tissues adjacent to peri-implant bone loss: expression of receptor activator NFkappaB, RANK ligand and osteoprotegerin.

Aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans. This study investigates the expression of key regulators of osteoclast formation, receptor activator NFkappaB (RANK), Receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin (OPG), in the peri-implant tissues of patients with osteolysis compared with levels in synovial tissues from osteoarthritic and healthy subjects. Immunohistochemical studies demonstrated that significantly higher levels of RANKL protein (p<0.05) were found in the peri-implant tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects. In contrast, OPG protein levels were similar in all tissues. RANKL, expressed as mRNA and protein, was predominantly associated with cells containing wear particles. Dual labeling studies showed that the cells expressing RANKL protein were macrophages. In situ hybridization studies confirmed that mRNA encoding for these proteins is also expressed by cells in the peri-implant tissues. In addition, RANK mRNA was expressed in cells that contained wear particles. These findings show that abnormally high levels of RANKL are expressed in peri-implant tissues of patients with prosthetic loosening and that these abnormal levels of RANKL may significantly contribute to aseptic implant loosening.

Adult↗

PubMed2003

Macrophages at the skeletal tissue-device interface of loosened prosthetic devices express bone-related genes and their products.

Aseptic loosening of prosthetic arthroplasty is the most common reason for implant failure in adult orthopaedic reconstruction. At the interface of aseptic loosened prostheses, there is an abundance of particle-activated macrophages and other inflammatory cells. The role of these particle-laden macrophages in the osteogenic arm of the remodeling skeleton in this pathological condition is poorly understood. Molecular signaling by mesenchymal cells and mononuclear inflammatory cells residing in the interfacial tissues between bone and cement or prosthetic material of aseptically loosened joint prostheses was studied using in situ hybridization and immunohistochemical techniques. We found that a range of collagenous and noncollagenous matrix proteins, including osteopontin, osteocalcin, bone sialoprotein, and type I collagen, were produced in the periprosthetic tissue by foamy macrophages, as well as nearby osteogenic cells. The former accumulated in profusion in the three zones of interfacial tissues: pseudomembranous, fibrous, and osseous. Spindle mesenchymal cells in the fibrous zone failed to express any of the osteogenic mRNAs or proteins sought. The expression of bone-related genes and proteins by foamy macrophages at the interface of an aseptic loosened prosthesis may contribute to the disturbance of bone remodeling at this site.

Adult↗

PubMed2003

Differentiation of human bone-derived cells grown on GRGDSP-peptide bound titanium surfaces.

Various surface modifications have been applied to titanium alloy (Ti-6Al-4V) implants, in an attempt to enhance osseointegration; crucial for ideal prosthetic fixation. Despite the numerous studies demonstrating that peptide-modified surfaces influence in vitro cellular behavior, there is relatively little data reporting their effects on bone remodeling. The objective of this article was to examine the effects of chemically modifying Ti-6Al-4V surfaces with a common RGD sequence, a 15-residue peptide containing GRGDSP (glycine-arginine-glycine-aspartate-serine-proline), on the modulation of bone remodeling. The expression of proteins known to be associated with osseous matrix and bone resorption were studied during the growth of human bone-derived cells (HBDC) on these peptide-modified surfaces. HBDC grown for 7 days on RGD surfaces displayed significantly increased levels of osteocalcin, and pro-collagen Ialpha1 mRNAs, compared with the production by HBDC grown on the native Ti-6Al-4V. A pattern that was also reflected at the protein levels for osteocalcin, type I collagen, and bone sialoprotein. Moreover, HBDC grown for 7 and 14 days on RGD-modified Ti-6Al-4V expressed significantly higher level of osteoclast differentiation factors and lower levels of osteoprotegerin and IL-6 proteins compared with other surfaces tested. These results suggest that different chemical treatments of implant material (Ti-6Al-4V) surface result in differential bone responses, not only their ability to form bone but also to stimulate osteoclastic formation.

Amino Acid Sequence↗

PubMed2003

Prosthetic particles modify the expression of bone-related proteins by human osteoblastic cells in vitro.

Loss of bone near joint prostheses is thought to be caused by activation of recruited osteoclasts by osteolytic mediators induced by wear particles. It is proposed that particles inhibit osteogenesis during bone remodelling causing a reduction in the levels of peri-implant bone. This study explores whether prosthetic particles modulate bone formation by affecting osteoblastic bone-related mRNAs (alkaline phosphatase, pro-collagen Ialpha1, osteopontin, osteonectin, osteocalcin, bone sialoprotein and thrombospondin) or their translated proteins using titanium alloy, commercially pure titanium, and cobalt-chrome particles. The direct effect of the particles revealed no change to the expression of the bone-related mRNAs in human bone-derived cells (HBDC) at the time points investigated; although non-collagenous translated proteins expressed by these HBDC were significantly effected (p<0.05). Different patterns of expression for bone-related proteins were induced by the different particles both directly and indirectly. Inflammatory mediators (interleukin-1beta, tumor necrosis factor alpha, interleukin-6, and prostaglandin E2) had similar effects on HBDC to the media obtained from monocytes incubated with particles. This study shows that prosthetic wear particles can significantly modify the expression of bone-related proteins by osteogenic cells in vitro. These alterations in osteogenic activity at the interface of the implant and bone may be an important factor in the failure of many orthopaedic implants.

Bone and Bones↗

PubMed2002

Mechanisms of magnesium-stimulated adhesion of osteoblastic cells to commonly used orthopaedic implants.

Poor cell adhesion to orthopaedic and dental implants may result in implant failure. Cellular adhesion to biomaterial surfaces primarily is mediated by integrins, which act as signal transduction and adhesion proteins. Because integrin function depends on divalent cations, we investigated the effect of magnesium ions modified bioceramic substrata (Al(2)O(3)-Mg(2+)) on human bone-derived cell (HBDC) adhesion, integrin expression, and activation of intracellular signalling molecules. Immunohistochemistry, flow cytometry, cell adhesion, cell adhesion blocking, and Western blotting assays were used. Our findings demonstrated that adhesion of HBDC to Al(2)O(3)-Mg(2+) was increased compared to on the Mg(2+)-free Al(2)O(3). Furthermore, HBDC adhesion decreased significantly when the fibronectin receptor alpha5beta1- and beta1-integrins were blocked by functional blocking antibodies. HBDC grown on the Mg(2+)-modified bioceramic expressed significantly enhanced levels of beta1-, alpha5beta1-, and alpha3beta1-integrins receptors compared to those grown on the native unmodified Al(2)O(3). Tyrosine phosphorylation of intracellular integrin-dependent signalling proteins as well as the expression of key signalling protein Shc isoforms (p46, p52, p66), focal adhesion kinase, and extracellular matrix protein collagen type I were significantly enhanced when HBDC were grown on Al(2)O(3)-Mg(2+) compared to the native Al(2)O(3). We conclude that cell adhesion to biomaterial surfaces is probably mediated by alpha5beta1- and beta1-integrin. Cation-promoted cell adhesion depends on 5beta1- and beta1-integrins associated signal transduction pathways involving the key signalling protein Shc and results also in enhanced gene expression of extracellular matrix proteins. Therefore, Mg(2+) supplementation of bioceramic substrata may be a promising way to improve integration of implants in orthopaedic and dental surgery.

Aluminum Oxide↗

PubMed2001

Preparation and analysis of macroporous TiO2 films on Ti surfaces for bone-tissue implants.

This article describes the preparation and analysis of macroporous TiO2 films on Ti surfaces, for application in bone tissue-Ti implant interfaces. These TiO2 bioceramic films have a macroporous structure consisting of monodisperse, three-dimensional, spherical, interconnected pores adjustable in the micron size range. Micron-sized polystyrene (PS) bead templates are used to precisely define the pore size, creating macroporous TiO2 films with 0.50, 16, and 50 microm diameter pores, as shown by scanning electron microscopy. X-ray photoelectron spectroscopy shows the films to be predominantly composed of TiO2, with approximately 10% carbon. X-ray diffraction reveal rutile as the main phase when fired to the optimal temperature of 950 degrees C. Preliminary experiments find that the in vitro proliferation of human bone-derived cells (HBDC) is similar on all three pore sizes. However, higher [3H]thymidine incorporation by the HBDC is observed when they are grown on 0.50- and 16-microm pores compared to the 50-microm pores, suggesting an enhanced cell proliferation for the smaller pores.

Biocompatible Materials↗

PubMed1999

Titanium substrata composition influences osteoblastic phenotype: In vitro study.

In spite of observed differences at the interface between boon and either commercially pure titanium [Ti(cpi)] or titanium alloy (Ti-6Al-4V), the mechanism of such a response is ill understood. This prompted further investigation of the influence of similar metals on human bone-derived cells (HBDCs). This study investigated the influence of Ti(cpi) and its alloy on osteoblastic proteins formed by HBDCs grown for 5, 7, 10, and 14 days on these metals and compared them to cells grown on tissue culture polystyrene plates. Messenger RNA and translated proteins that form an array of osteogenic parameters were determined: alkaline phosphatase (ALP), thrombospondin, osteopontin, osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I) and bone sialoprotein (BSP). At the four predetermined time points, cells grown on either Ti(cpi) or Ti-6Al-4V generally expressed similar mRNA levels, while levels of their respective proteins differed. Cells on Ti(cpi) had peak levels for most proteins at day 7, whereas those on Ti-6Al-4V peaked at either day 5 and/or day 7. At day 5 cells grown on Ti-6Al-4V had higher levels of ALP, Col I, ON/SPARC, OC, and BSP than those in Ti(cpi); this difference was not maintained at later time points in culture. The differential regulation of proteins occurring between cells from the same patient grown on titanium and its alloy implies that HBDCs respond to small differences in the surface chemistry and/or microcrystallinity.

Adolescent↗

PubMed1999

Effect of ion modification of commonly used orthopedic materials on the attachment of human bone-derived cells.

Biomaterials which combine optimum properties of strength and biocompatibility are desirable in improving the long-term performance of implantable medical devices. Our study is aimed at developing technology designed to alter the outer atomic layers of a material to give the desired compatibility with the tissue while retaining the properties of the bulk substratum. Materials used in this study were titanium vanadium alloy (Ti-6Al-4V) and cobalt chromium molybdenum alloy (Co-Cr). Soda lime glass discs and polyethylene terephthalate (PET) acted as controls. A cathode of either Ti-6Al-4V or Co-Cr was used to simultaneously deposit and implant identified substrata. The attachment of human bone-derived cells (HBDC) to various materials was determined using radiolabeling or colorimetric assays. Results show that HBDC adhere preferentially to the unmodified surfaces of Ti-6Al-4V and Ti-6Al-4V on glass compared to the unmodified Co-Cr surfaces and to that of the Co-Cr on glass. Depositing Ti-6Al-4V on Co-Cr gives significantly better attachment of HBDC than when depositing Co-Cr onto Ti-6Al-4V. While cellular attachment to the created surfaces reflects that of the cathodic materials, it is not identical to these materials. Ion deposition/implantation is capable of creating permanent surfaces which reflect the adhesion of source materials not bulk substrata.

Alloys↗

PubMed1999

Effect of surface chemical modification of bioceramic on phenotype of human bone-derived cells.

In the search for methods to improve the biocompatibility of prosthetic materials, attention has recently been directed toward the potential use of surface chemical modification and its influence on cellular behavior. This in vitro study investigates the effect of surface chemistry modification of bioceramics on human bone-derived cells (HBDCs) grown on biomaterial surfaces for 2 weeks. Cells were cultured on either alumina (Al2O3), alumina doped with magnesium ions ([Mg]-Al2O3), or hydroxyapatite (HAP), as well as tissue culture polystyrene (TCPS). Expression of alkaline phosphatase (ALP), thrombospondin (Tsp), osteopontin (OP), osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I), and bone sialoprotein (BSP) were determined in terms of mRNAs and proteins. Protein levels for ALP, OP, OC, and BSP were significantly (p < 0. 05) greater at day 5 in HBDCs cultured on [Mg]-Al2O3 compared to those cells grown on Al2O3. At day 14 the levels of ALP, Tsp, Col I, OP, ON/SPARC, and BSP rose significantly (p < 0.05) above those occurring in HBDCs grown on Al2O3, HAP, and TCPS. This suggests that HBDCs from the same patient respond to differences in the surface chemical groups. This study confirms that the chemistry of a substratum, which facilitates cellular adhesion, will enhance cellular differentiation.

Adolescent↗

PubMed1999

The effect of polymeric chemistry on the expression of bone-related mRNAs and proteins by human bone-derived cells in vitro.

This study used human bone-derived cells (HBDC) grown on two defined polymeric substrata to examine the effect of substrata chemistry on the expression of mRNAs and proteins characteristic of the osteoblastic phenotype. The growth profile of cells grown on tissue culture polystyrene (TCP) was exponential whereas for those seeded on polyethyleneterephthalate (PET) there was a pronounced lag period before cellular multiplication. The temporal expression pattern of mRNAs in HBDC cultured on TCP was similar to that of cells on PET. On TCP, the levels of several mRNAs peaked at day 4, as cellular proliferation slowed. In contrast, the induction in mRNA levels in cells grown on PET corresponded to maximum mitotic activity. There appears to be sequential cascade in protein expression in cells grown on TCP with overlapping peaks of thrombospondin (Tsp), osteocalcin (OC) and osteopontin (OP) expression. In contrast, peak intracellular protein expression levels for Tsp, OC and OP did not overlap when cells were grown on PET.

Adolescent↗

PubMed1998

Quantitative aspects of an in situ hybridization procedure for detecting mRNAs in cells using 96-well microplates.

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitative in situ hybridization (ISH) using either image analysis or fluorescence in situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by the p-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.

Animals↗

PubMed1998

Quantification of the bone-related mRNAs at the bone/prosthetic interface.

Aseptic loosening is one of the major reasons for failure of joint prostheses. The periprosthetic tissue has previously been described microscopically; however, little work has been devoted towards quantitating genes expressed by cells at the materials/tissue interface. This study aims to characterize the phenotypic expression of osteoblasts and test the feasibility of quantifying the level of gene expression in periprosthetic tissue sections by combining in situ hybridization and image analysis techniques. There are many factors to consider when quantifying mRNA, in that comparing labeling between different cDNA probes, these should have comparable length and base comparison. The probes should be labeled with the same specific activity, that is the amount of probe to label added is the same, both between different probes and between batches of the same probe. Chromagen color reactions are variable in that the color development is not always linear and more likely follows a sigmoidal curve. Samples should only be compared when it is known that the reaction has been in the linear range. The image analysis of such staining introduces further factors which should be considered and controlled. Color analysis is a very complex problem with respect to reproducibly analyzing histological sections. The brightness component of the image should be independent of the colors within the image, in conventional RGB (red, green and blue) signalling mode this is not possible, while when using HSI (hue saturation and intensity) mode this becomes possible, and factors like staining intensity and brightness of the image become much more accountable and controllable. With these factors identified, we consider that the quantitative image analysis approach does allow comparison of patterns of bone-related mRNAs and demonstrates differences in expression in these osteogenic factors depending on distance from the prosthesis, tissue type, patient and device.

Journal Article↗