Histological evaluation of repair of the rotator cuff in a primate model.

The establishment of a suitable animal model of repair of the rotator cuff is difficult since the presence of a true rotator cuff anatomically appears to be restricted almost exclusively to advanced primates. Our observational study describes the healing process after repair of the cuff in a primate model. Lesions were prepared and repaired in eight 'middle-aged' baboons. Two each were killed at four, eight, 12 and 15 weeks post-operatively. The bone-tendon repair zones were assessed macroscopically and histologically. Healing of the baboon supraspinatus involved a sequence of stages resulting in the reestablishment of the bone-tendon junction. It was not uniform and occurred more rapidly at the sites of suture fixation than between them. Four weeks after repair the bone-tendon healing was immature. Whereas macroscopically the repair appeared to be healed at eight weeks, the Sharpey fibres holding the repair together did not appear in any considerable number before 12 weeks. By 15 weeks, the bone-tendon junction was almost, but not quite mature. Our results support the use of a post-operative rehabilitation programme in man which protects the surgical repair for at least 12 to 15 weeks in order to allow maturation of tendon-to-bone healing.

S100A8/S100A9 and their association with cartilage and bone.

S100A8 and S100A9 are calcium-binding proteins expressed in myeloid cells and are markers of numerous inflammatory diseases in humans. S100A9 has been associated with dystrophic calcification in human atherosclerosis. Here we demonstrate S100A8 and S100A9 expression in murine and human bone and cartilage cells. Only S100A8 was seen in preosteogenic cells whereas osteoblasts had variable, but generally weak expression of both proteins. In keeping with their reported high-mRNA expression, S100A8 and S100A9 were prominent in osteoclasts. S100A8 was expressed in alkaline phosphatase-positive hypertrophic chondrocytes, but not in proliferating chondrocytes within the growth plate where the cartilaginous matrix was calcifying. S100A9 was only evident in the invading vascular osteogenic tissue penetrating the degenerating chondrocytic zone adjacent to the primary spongiosa, where S100A8 was also expressed. Whilst, S100A8 has been shown to be associated with osteoblast differentiation, both S100A8 and S100A9 may contribute to calcification of the cartilage matrix and its replacement with trabecular bone, and to regulation of redox in bone resorption.

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.

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.

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.

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.

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.

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.

Productive infection of the bone marrow cells in feline immunodeficiency virus infected cats.

Bone marrow stromal cells (BMSC) from cats experimentally infected with feline immunodeficiency virus (FIV) were shown to contain FIV provirus using polymerase chain reaction and viral products were detected in culture supernatant using reverse transcriptase and enzyme linked immunosorbent assay techniques. Peripheral blood mononuclear cells from FIV-free cats co-cultured with infected bone marrow cells became productively infected with FIV. Such evidence supports the hypothesis that BMSC are a reservoir for FIV. Furthermore, BMSC produced virions capable of infecting susceptible cells and may represent an important source of infectious virus to cells of the macrophage lineage and/or hemopoietic progenitor cells, both of which ultimately become widely disseminated throughout the body.

Effects of glass ionomer cements on bone tissue.

In vivo biocompatibility of glass ionomer cements (GICs) was evaluated for use in orthopaedic surgery using a rat model and compared with conventional bone cement, Polymethyl methacrylate, PMMA. The unset GICs and PMMA were inserted into the marrow cavities of rat femora and retained in situ for various periods of time. The PMMA bone cement showed complete biocompatibility with no interference with reparative bone. The conventional GIC with smaller glass particles and lower powder/liquid ratio showed an initial minor toxic effect on rat bone tissue with later disturbance of adjacent bone formation. The conventional GIC with larger-size glass particles and higher powder/liquid ratio and resin-modified GIC showed more severe toxic effect on rat tissue with the resin-modified GIC affecting the rat bone tissue later. The causes of toxicity associated with the conventional GIC with larger glass particles and higher powder/liquid ration and the resin-modified GIC are thought to be related with the unreacted acid component of both materials and longer ongoing metallic ion release.

Laminated tears of the human rotator cuff: a histologic and immunochemical study.

Laminated tears of the rotator cuff are often lined by a cellular layer that has an appearance suggestive of synovium. This study demonstrates, by histologic and immunohistochemical means, that the lining cells are synovial. It remains unclear whether these cells arise by synovial extension from the joint or bursa, or by metaplasia in the presence of synovial fluid, and this has implications for surgical repair of laminated cuff defects. We suggest that these defects be curetted, to remove at least some of this synovial lining, before suture repair.

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.

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.

Nitrous acid pretreatment of tendon xenografts cross-linked with glutaraldehyde and sterilized with gamma irradiation.

Collagenous xenografts made from kangaroo tail tendon cross-linked with glutaraldehyde have a potential application in the reconstruction of massive digital tendon deficits. However, a limitation to the clinical use of these xenografts has been the optimization of collagen cross-linking, and subsequent bio-incorporation and retention of mechanical properties following implantation. The purpose of this study was to evaluate the effect of nitrous acid on modulating the biologic and mechanical properties of tendon xenografts cross-linked with glutaraldehyde. Tendon xenografts were pretreated with 0.1 or 0.01 M nitrous acid solution, prior to cross-linking in 2% glutaraldehyde and sterilization by gamma irradiation. Xenografts were implanted intramuscularly in rabbits to examine biocompatability, and also used to repair ovine digital extensor tendon deficits to evaluate functional incorporation. Histologically, intramuscularly implanted nitrous acid pretreated xenografts in rabbits had a greater degree of diffuse cellular infiltration into interstitial splits in the graft than controls after 12 weeks. Xenografts implanted in an ovine extensor tendon deficit were evaluated after 26 and 52 weeks. Rate of failure of tenorrhaphies between host tendon and xenografts overall (15/21) was significantly greater (P < 0.05) than for autografts (1/21), suggesting that the holding power of sutures in xenografts was inferior to that obtained in autografts. Tensile failure stress of midsections of both nitrous acid pretreated and control xenografts was about 100 MPa prior to implantation (time zero). After 26 and 52 weeks, failure stress of both types of xenografts was significantly less than at time zero (P < 0.05). At 52 weeks, failure stress of nitrous acid pretreated xenografts (47.4 +/- 3.1 MPa) was significantly less than control xenografts (63.7 +/- 5.4 MPa); (P < 0.05). However, nitrous acid pretreated xenografts were similar to control xenografts in failure load (357 +/- 29 and 354 +/- 26 N, respectively), but they tended to have larger cross-sectional areas (7.6 +/- 0.5 versus 5.7 +/- 0.6 mm2, respectively) which were responsible for the lower calculated value for failure stress. Histologically, autografts maintained their normal tissue architecture and evoked a more limited cellular response in surrounding tissues than xenografts (P < 0.05). Both types of xenograft were surrounded by a thicker cuff of cellular response than autografts. However, compared to control xenografts, nitrous acid pretreated xenografts had more extensive fragmentation and splitting of collagen bundles, and more diffuse cellular and vascular infiltration into these interstitial splits, and these alterations were apparently contributing to the greater 'swelling' of these xenografts. It was concluded that pretreatment of tendon xenografts with nitrous acid modulated their biologic and material properties. Further studies are needed to elucidate the mechanism of these effects, and to determine if the protocol for tendon xenograft preparation could be optimized for improved clinical performance.

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.

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.

Osteochondrodysplasia in Scottish Fold cats.

OBJECTIVE: To better characterise the bone and joint problems which can develop in Scottish Fold cats. DESIGN: Retrospective study of cases seen in five veterinary clinics and radiographic survey of cats in a cattery. RESULTS: Six Scottish Fold cats (four castrated males, two spayed females) aged between 5 months and 6 years were presented for signs of skeletal disease including lameness, reluctance to jump, a stiff stilted gait, short misshapen distal limbs, swelling of plantar tarsometatarsal regions and short thick inflexible tails. A further four cases (one male, three females, 15 months to 11 years) were identified by radiographic screening of a cattery. A diagnosis of osteochondrodysplasia was based on characteristic radiological findings including irregularity in the size and shape of tarsal, carpal, metatarsal and metacarpal bones, phalanges and caudal vertebrae, narrowed joint spaces, and progressive new bone formation around joints of distal limbs with diffuse osteopenia of adjacent bone. A plantar exostosis caudal to the calcaneus was present in advanced cases. In all nine cases where pedigree information was available, affected cats allegedly originated from the mating of a Scottish Fold to a cat with normal ears. The severity and time of onset of physical signs, and rate of progression and extent of radiographic abnormalities, varied from case to case. Limited histological observations suggested the underlying problem may be an osteochondrodysplasia, related to inadequate cartilage maturation. Clinical signs were ameliorated by administration of pentosan subcutaneously in two of three cats in which it was trailed, and one of these also benefited from an oral glycosaminoglycan preparation. CONCLUSIONS: Clinical and radiological findings were ascribed to defective maturation and function of cartilage, particularly in the distal limbs, ears and tail. As all Scottish Fold cats suffered from osteochondrodysplasia of some degree, the best solution would be to avoid using fold-eared cats for breeding and instead use Scottish shorthairs.

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.

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.