3D cell culture studies more accurately represent the complex in vivo mechanical environment of human bone and are, thus, superior to 2D studies when testing the efficacy of osteoporosis therapies. As such, the objective of this study was to use a 3D model to investigate the effect of sclerostin antibodies. Sclerostin is a protein, which inhibits osteoblasts and is downregulated under mechanical stimulation. It is not yet known how expression of sclerostin mediates the site-specific and temporal changes in mineralisation. To address this, we developed a 3D cellular niche of MC3T3 osteoblasts encapsulated within gelatin and applied mechanical loading to the constructs using a custom-designed compression bioreactor system (0.5% strain at 0.5 Hz, 1 hr/day) (VizStim) under continuous perfusion of cell culture media. Osteoblasts were pretreated with estrogen for 14 days, followed by estrogen withdrawal (EW) to simulate postmenopausal conditions. 3D constructs were successfully fabricated and actin staining revealed the formation of dendritic cells under both static and stimulated conditions indicative of osteocyte-like cells. Under static conditions, estrogen treatment enhanced production of calcium by osteoblasts when compared to the same cells cultured under estrogen deficient conditions. Overall, preliminary results propose a link between mechanical stimulation, estrogen deficiency and mineral production by osteoblasts. Ongoing studies are comparing the static and stimulated groups after a longer culture period of 21 days using sclerostin antibodies. This research aims to deliver further understanding of the mechanical regulation of bone formation, and will inform novel approaches for regeneration of bone tissue and treatment of osteoporosis

Osteoporosis has long been associated with weak bones but recent studies have shown that bone tissue mineral becomes more heterogeneous and the expression of mechanosensors are altered during estrogen deficiency in an animal model of osteoporosis. However, whether these changes occur as a primary response to estrogen deficiency is unknown. In this study we investigate whether matrix production and mineralisation by mechanically-stimulated osteoblasts are impaired as a direct consequence of estrogen depletion. Osteoblast-like MC3T3-E1 cells were cultured for 14 days with 10. −8. M of 17β-estradiol and subsequently cultured with osteogenic media only, or supplemented with estrogen or an estrogen antagonist (Fulvestrant, 10. −7. M). Physiological shear stress (1Pa) was applied using an orbital shaker (290rpm, 40min/day), which allows long-term culture and induces oscillatory flow on cells. Osteoblasts phenotype, extracellular matrix (ECM), mineralisation and mechanosensors were tracked by qRT-PCR (Runx2, Col1a1, Col1a2, Cox2, Bglap2, FN1), by biochemical assays (ALP activity, DNA and calcium content), by immunostaining (integrin α. v. , BSP2, fibronectin) and by labelling with calcein the calcium. The results of this study demonstrate that after 7 days, estrogen depleted cells had less integrin α. v. mechanosensors compared to those that received continuous estrogen treatment. By 14 days the ECM formation (calcium, fibronectin) by osteoblasts was altered under estrogen depletion, when compared to cells that were cultured continuously with estrogen. This study provides evidence of changes in osteoblast behaviour under estrogen depletion, which might explain the alteration in tissue mineral content and the decrease of integrins observed previously in ovariectomized rats in vivo

Complement C5a receptor 1 (C5aR1) has crucial functions in host defense against danger molecules, as does toll-like receptor 2 (TLR2). Both innate immunity receptors interact in immune cells in the context of infectious inflammatory diseases often associated with bone loss, such as periodontitis. C5aR1 plays an important role in bone, as it is expressed on bone cells and strongly upregulated due to bone injury. Importantly, C5aR1-ko mice are protected against arthritis and C5aR1 contributes to bone loss in periodontitis. In contrast, less data exist on the role of TLR2 on osteoblasts, however, it is known that TLR2 is expressed on osteoblasts and contributes to bacterial-induced bone resorption. The aim of this study was to evaluate the interaction of C5aR1 and TLR2 in osteoblasts, including intracellular signaling pathways and gene expression patterns. Primary osteoblasts were isolated from 8–12 week-old WT mice and differentiated for 14 days. Osteoblasts were assessed for expression of C5aR1 and TLR2. Phosphorylation of mitogen-activated protein kinases (MAPK) in response to C5a and Pam3CSK4 (TLR2 agonist) was analyzed by immunoblotting. Gene expression profiling after 30 min and 4 h stimulation of C5a was performed by microarray and candidate genes were validated by quantitative Real-Time PCR (qRT-PCR). Immunoprecipitation was performed using a C5aR1-antibody and C5aR1 and TLR2 were subsequently detected by immunoblotting. Statistics: One way ANOVA p<0.05, n=4–6. We showed that C5aR1 and TLR2 are expressed on osteoblasts and strongly upregulated during differentiation. Via immunoprecipitation, we could show that C5aR1 and TLR2 do physically interact in osteoblasts. We then examined if C5aR1 and TLR2, besides their physical interaction, also act via the same intracellular signaling pathways. Gene expression profiling upon C5a stimulation revealed that the top regulated pathways are related to MAPK and transforming growth factor beta (TGF-β). Respective genes, such as TGF-β (Tgfb1) and its receptor (Tgfbr) were found to be upregulated, and negative MAPK regulators were found to be downregulated, both by microarray analysis and qRT-PCR. Accordingly, we saw a C5aR1- and TLR2-dependent phosphorylation of p38 MAPK. Interestingly, this effect was enhanced and prolonged by costimulation of both receptors. An additive effect of C5aR1 and TLR2 was also seen regarding Cxcl10 levels, which were enhanced compared to C5aR1 or TLR2 stimulation alone. This study shows that C5aR1 and TLR2 interact in osteoblasts, not only physically but also functionally, regarding downstream signaling and target genes. Those data strongly imply a synergistic interplay between the receptors, through which osteoblasts possibly contribute to inflammatory reactions affecting bone

We used an atomic layer deposition (ALD) approach to create titanium oxide nanolayers on ultra high molecular weight polyethylene (UHMWPE) surfaces. These materials were then characterised in terms of rat osteoblast adhesion, morphology and differentiation. UHMWPE discs produced from a machined cylinder or impact moulded discs were coated with titanium oxide by ALD. Light, atomic force microscopy and scanning electron microscopy with EDX were used to characterise the coated surfaces. These approaches showed 1-1.5 micron tooling grooves with a periodicity of 40 microns on the machined discs whilst the moulded discs exhibited nanotopographical features. The titanium oxide coating was successfully deposited on discs from both sources but was not uniform across the surfaces, with vein-like ‘creases’ clearly visible. We believe that these features are due to the thermal expansion of the UHMWPE discs during the ALD process and their subsequent cooling. Coated and uncoated discs were seeded with osteoblasts for 24 hours, then fixed. Immunofluorescence microscopy and computer-based image processing enabled determination of osteoblast numbers, size and shape. A trend of larger average cell area was associated with the coated discs and P<0.01 for an H0 of no difference in cell area between coated and uncoated grooved discs. Osteoblasts were also cultured on the discs in osteogenic medium to promote bone nodule formation. After a few weeks, von Kossa staining and computer-based image processing allowed calculation of surface area covered with bone nodules for each of the discs. Based on results from three of each type of disc, a significantly greater proportion of the surface area of coated discs was covered with calcified deposits compared to uncoated discs (P<0.025 for grooved discs and P<0.005 for smooth discs). On average, the coated discs had bone nodules on 1.4 times the surface area as compared to their uncoated counterparts. The hypothesis for our study was that TiO2 coating of a polymer might better promote osteoblast interaction with the biomaterial surface leading to enhanced osteogenesis. Our preliminary data support this view and suggest that this approach could likely be exploited in the fabrication of implant materials with tailored biological activity

Total hip replacement (THR) is indicated for patients with osteoarthritis where conservative treatment has failed. Metal alloys used in THR implants such as cobalt-chromium (CoCr) have been known to cause pro-inflammatory reactions in patients, therefore leading to the need for costly revision surgery. This study therefore aimed to investigate the role of TLR4 in the activation of a human osteoblast model in response to CoCr particles in vitro.

Human osteoblasts (MG-63 cell line) were seeded at a density of 100,000 cells and treated with 0.5, 5, 50mm3 CoCr particles per cell for 24-hours. Trypan blue and the XTT Cell Proliferation Kit II were then used in conjunction with the cells to assess CoCr-induced cytotoxicity. Cells were pre-treated with a commercially available TLR4-specific small molecule inhibitor (CLI-095) for 6 hours. Untreated cells were used as a negative control and lipopolysaccharide (LPS) was used as a positive control. Following treatment the cell supernatant was collected and used for enzyme-linked immunosorbant assay (ELISA) to measure the secretion of interleukin-8 (IL-8), CXCL10, and interleukin-6 (IL-6).

Trypan blue and XTT analysis showed that there was no significant changes to cell viability or proliferation at any dose used of CoCr after 24 hours. There was a significant increase in protein secretion of IL-8 (p<0.001), CXCL10 (p<0.001), and IL-6 (p<0.001) in the cells which received the highest dosage of CoCr. This pro-inflammatory secretory response was ameliorated by TLR4 blockade (p<0.001).

CoCr particles are not cytotoxic to osteoblasts but they do induce pro-inflammatory changes as characterised by increased secretion of chemokines IL-8, CXCL10, and IL-6. These responses occur via a TLR4-mediated pathway and upon inhibition they can be effectively ameliorated. This is particularly important as TLR4 could be a potential target for pharmacological intervention used in patients experiencing immunological reactions to metal implant debris.

Introduction and Objective

Mesenchymal stem cells (MSC) are attractive candidates for bone regeneration approaches. Benefits of MSC therapy are mainly attributed to paracrine effects via soluble factors, exerting both immunoregulatory and regenerative actions. Encapsulation of MSC in hydrogels prepared with extracellular matrix (ECM) proteins has been proposed as a strategy to enhance their survival and potentiate their function after implantation. Functional activity of MSC can be regulated by the physical and mechanical properties of their microenvironment. In this work, we investigated whether matrix stiffness can modulate the crosstalk between MSC encapsulated in collagen hydrogels with macrophages and osteoblasts.

The increased incidence of type 2 Diabetes Mellitus is associated with an impaired skeletal structure and a higher prevalence of bone fractures. Sclerostin is a negative regulator of bone formation produced by osteocytes and there is recent evidence that its expression in serum is elevated in diabetic patients compared to control subjects. In this study, we test whether hyperglycemia affects serum and bone sclerostin levels in a rat model of type 2 Diabetes as well as sclerostin production by osteoblasts in culture.

We used Zucker diabetic fatty (ZDF) male rats (n=6) that spontaneously develop obesity and frank diabetes around 8–9 weeks of age and Zucker lean rats as controls (n=6) to examine sclerostin expression in serum at 9, 11 and 13 weeks using a specific ELISA. Sclerostin expression in bone tibiae was examined at 12 weeks using immunocytochemistry. Rat osteoblast-like cells UMR-106 were cultured in the presence of increasing concentrations of glucose (5, 11, 22 and 44 mM) during 48 hours and sclerostin mRNA expression and release in the supernatant determined by quantitative PCR and ELISA, respectively.

Our results show that serum sclerostin levels are higher in the diabetic rats compared to lean rats at 9 weeks (+ 140%, p<0.01). Our preliminary results using immunocytochemistry for sclerostin did not show any major difference in sclerostin expression in tibiae of diabetic rats compared to lean ones, although we observed many osteocytic empty lacunae in cortical bone from diabetic rats. Glucose dose-dependent stimulated sclerostin mRNA and protein production in mature UMR106 cells while it had no effect on osteocalcin expression.

Altogether, our data suggest that sclerostin production by mature osteoblasts is increased by hyperglycemia in vitro and enhanced in serum of diabetic rats. Furthers studies are required to determine whether sclerostin could contribute to the deleterious effect of Diabetes on bone.

Bone related adverse events including failure of implant osseo-integration, periprosthetic fracture, femoral neck narrowing, and unexplained pain occur more frequently following metal-on-metal hip resurfacing (MoMHR) versus total hip arthroplasty (THA). The exact mechanism for the adverse effects is still unclear and may be due to the direct effect on bone cells of metal ions released from the prostheses.

The aim of the present study was to determine the effect of clinically relevant combinations of metal ions on osteoblast cell survival and function. To assess cell proliferation and alkaline phosphatase (ALP) activity of osteoblasts, human osteoblast cells (SaOS-2), were cultured in 96-well plates for 24-hours and then treated with metal ions. Cell proliferation was measured at day 3 and day 7 using MTS assay, whilst ALP activity was assessed at day 3 by measuring pNPP substrate hydrolysis by the cell lysate. Mineralisation ability of the cells was assessed in 24-well plates cultured until day 21 and staining the calcium deposits using Alizarin red. All cultures were treated with the IC50 concentration of Co(II) (135μM) and an equivalent Cr(III) concentration (1Co(II):1Cr(III)).

After 3 days, Co(II) at an IC50 concentration decreased osteoblast proliferation as expected, but no further decrease in proliferation was observed with the 1Co(II):1Cr(III) combination treatment. However, after 7 days, a further significant decrease (P<0.05) in proliferation was observed with the combination treatment compared to Co(II) IC50. A similar significant decrease (P<0.01) was observed for ALP activity at day 3 with 1Co(II):1Cr(III) compared to Co(II) alone. For mineralization, a significant reduction (P<0.0001) was observed for Co(II) IC50 concentration, however no further reduction was seen with the 1Co(II):1Cr(III) combination treatment.

The observed decrease in cell proliferation and ALP activity with combination treatments suggest an additive detrimental effect compared to single ions alone. The mineralisation ability did not show any additive effect due to cell toxicity of chronic exposure to IC50 concentrations calculated from 3 day proliferation cultures. The results suggest that presence of both cobalt and chromium ions in the periprosthetic environment have more severe detrimental effect on osteoblasts than single ions alone and extend our understanding of the periprosthetic bone health.

Introduction

Migration of bone cells and precursor cells to the site of a bone defect can accelerate bone regeneration. Therefore, guidance of these cells by direct current (DC) is an interesting approach to improve implant ingrowth or fracture healing. To allow a better understanding of DC-induced directed migration, a specific stimulation chamber was established and the influence of DC on calcium channel expression in osteoblasts was investigated.

Commonly used alterations of prosthetic surfaces include grit-blasting (GB), plasma-sprayed titanium (Ti) or hydroxyapatite (HA) coating. Systemic concentrations of cobalt (Co) and chromium (Cr) are elevated in patients with metal-on-metal hip replacement, but can occur for all modular hip replacements. Here, we use whole genome microarrays to assess differential gene expression in primary human osteoblasts grown in vitro and on these prosthesis surfaces following exposure to clinically relevant concentrations of Co and Cr.

Mesenchymal cells obtained from bone-fragments of 3 patients undergoing joint replacement surgery were differentiated into osteoblasts. Subsequently, cells were cultured in vitro on tissue-culture plates (TCP), or on GB, Ti and HA surfaces (JRI Orthopaedics Ltd, Sheffield, UK). Following 24hr exposure to a combination of clinically equivalent concentrations of Co2+:Cr3+, RNA was extracted and hybridized to SurePrint-G3 Gene Expression Microarray. Probe signals were normalised using ‘Limma’ package on R-Bioconductor and differential gene expression assessed with empirical Bayes approach (Log2FC>1.00, P<0.001 for differentially expressed genes).

For cells grown on TCP, 11 genes were upregulated with 500μg/L Co2+:Cr3+. Of these, 4 were associated to HIF-1 signalling based on KEGG pathway analysis (P=5.4e-5). Exposure to 1000μg/L Co2+:Cr3+ altered expression at 164 loci for HA surfaces, and a separate 50 loci for Ti surfaces compared to GB surfaces. Genes for osteoblast differentiation (BMP2 and RGS2) were downregulated on HA surfaces compared to GB, whilst genes for cell-adhesion (ESAM), vesicular trafficking (RAB37) and protection against oxidative damage (NRF2) were upregulated. Ti surfaces caused an upregulation in ERBB3 and CNTF, which are associated with inhibition of osteoblast differentiation and mineralisation, when compared to GB surfaces.

This study confirms the role of HIF-1 signalling in response to prosthesis generated metal ions, and is the first to provide a comprehensive genome-wide insight into transcriptional response of osteoblasts at prosthesis surface to clinically equivalent metal exposure.

Introduction

Despite the high regenerative capacity of bone, large bone defects often require treatment involving bone grafts. Conventional autografting and allografting treatments have disadvantages, such as donor site morbidity, immunogenicity and lack of donor material. Bone tissue engineering offers the potential to achieve major advances in the development of alternative bone grafts by exploiting the bone-forming capacity of osteoblastic cells. However, viable cell culture models are essential to investigate osteoblast behavior. Three-dimensional (3D) cell culture systems have become increasingly popular because biological relevance of 3D cultures may exceed that of cell monolayers (2D) grown in standard tissue culture. However, only few direct comparisons between 2D and 3D models have been published. Therefore, we performed a pilot study comparing 2D and 3D culture models of primary human osteoblasts with regard to expression of transcription factors RUNX2 and osterix as well as osteogenic differentiation.

Objectives

Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought.

Objectives

Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown.

Introduction

Parathyroid hormone-related peptide (PTHrP) has been shown to be an important regulator of bone remodelling1. The aim of this study was to investigate the effect of the N-terminal domain of PTHrP (1–36) on osteogenic and angiogenic gene expression in human osteoblasts (HOB) and human bone marrow stromal cells (hBMSCs).

Introduction and Objective. Osteonecrosis of the femoral head (ONFH) is an evolving and disabling condition that often leads to subchondral collapse in late stages. It is the underlying diagnosis for approximately 3%–12% of total hip arthroplasties (THAs) and the most frequent aetiology for young patients undergoing THA. To date, the pathophysiological mechanisms underlying ONFH remain poorly understood. In this study, we investigated whether ONFH without an obvious etiological factor is related to impaired osteoblast activities, as compared to age-matched patients with primary OA. Materials and Methods. We cultured osteoblasts isolated from trabecular bone explants taken from the femoral head of patients with ONFH and from intertrochanteric region of patients with ONFH or with OA and compared their in vitro mineralisation capacity and secretion of paracrine factors. Results. Compared to patients with OA, osteoblasts obtained from the intertrochanteric region of patients with ONFH showed reduced mineralisation capacity, which further decreased in osteoblasts from the femoral head of the same patient. Lower mineralisation of osteoblasts from patients with ONFH correlated with lower mRNA levels of genes encoding osteocalcin and bone sialoprotein and higher osteopontin expression. Osteoblasts from the intertrochanteric region of patients with ONFH secreted lower osteoprtegerin levels than those from patients with OA, resulting in a higher receptor activator of NF-κB ligand (RANKL)-to-osteoprotegerin (OPG) ratio. Notably, the RANKL-to-OPG ratio, as well as the secretion of the proresorptive factors interleukin-6 and prostaglandin E. 2. , was higher in osteoblasts from the femoral head of patients with ONFH than in those from the intertrochanteric region. Conclusions. ONFH is associated with a reduced mineralisation capacity of osteoblasts and increased secretion of proresorptive factors

Several electrical fields are known to be present in bone tissue as originally described by Fukada and Yasuda in the year 1957. Intrinsic voltages can derive from bone deformation and reversely lead to mechanical modifications, called the piezoelectric effect. This effect is used in the clinic for the treatment of bone defects by applying electric and magnetic stimulation directly to the bone supplied with an implant such as the electroinductive screw system. Through this system a sinusoidal alternating voltage with a maximum of 700 mV can be applied which leads to an electric field of 5–70 V/m in the surrounding bone. This approach is established for bone healing therapies. Despite the established clinical application of electrical stimulation in bone, the fundamental processes acting during this stimulation are still poorly understood. A better understanding of the influence of electric fields on cells involved in bone formation is important to improve therapy and clinical success. To study the impact of electrical fields on bone cells in vitro, Ti6Al4V electrodes were designed according to the pattern of the ASNIS III s screw for a 6-well system. Osteoblasts were seeded on collagen coated coverslip and placed centred on the bottom of each well. During four weeks the cells were stimulated 3×45 min/d and metabolic and alkaline phosphatase (ALP) activity as well as gene expression of cells were analysed. Furthermore, supernatants were collected and proteins typical for bone remodelling were examined. The electrical stimulation did not exert a significant influence on the metabolic activity and the ALP production in cells over time using these settings. Gene expression of BSP and ALP was upregulated after the first 3 days whereas OPG was increased in the second half after 14 days of electrical stimulation. Moreover, the concentration of the released proteins OPG, IL-6, DKK-1 and OPN increased when cells were cultivated under electrical stimulation. However, no changes could be seen for essential markers, like RANKL, Leptin, BMP-2, IL-1beta and TNF-alpha. Therefore, further studies will be done with osteoblasts and osteoclasts to study bone remodelling processes under the influence of electrical fields more in detail. This study was supported by the German Research Foundation (DFG) JO 1483/1-1

High-pressure lavage produces greater visible damage to bone at a macroscopic and microscopic level when compared with low-pressure lavage and can result in delay in the healing of fractures. Osteoblasts and adipocytes are derived from mesenchymal stem cells. Conditions which lead to bone loss often involve a switch from the osteoblast to adipocyte lineage. We have therefore examined the effect of high- and low-pressure irrigation on the differentiation of adipocytes. Calvaria-derived bone cells were exposed to either low-pressure or high-pressure irrigation with normal saline. After 14 days the cells were fixed and the osteoblasts and adipocytes quantified using Oil Red O to stain cytoplasmic lipid droplets (triglycerides) in the cells. Osteoblasts were quantified using a commercially available alkaline-phosphatase staining assay. A standard quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed. Messenger RNA levels for osteocalcin, a marker of osteoblasts, and PPARγ2, a marker of adipocytes, were measured. High-pressure lavage resulted in an increase in adipogenesis of 50% when compared with low-pressure lavage. Our findings suggest that high-pressure lavage may promote differentiation of mesenchymal stem cells towards the adipoctye lineage. This may have clinical significance in the development of delayed and nonunion after treatment of fractures of long bones

We have examined the process of fusion of the intertransverse processes and bone graft in the rabbit by in situ hybridisation and evaluated the spatial and temporal expression of genes encoding pro-α1 (I) collagen (COL1A1), pro-α1 (II) collagen (COL2A1) and pro-α1 (X) collagen (COL10A1). Beginning at two weeks after operation, osteogenesis and chondrogenesis occurred around the transverse process and the grafted bone at the central portion of the area of the fusion mass. Osteoblasts and osteocytes at the newly-formed woven bone expressed COL1A1. At the cartilage, most chondrocytes expressed COL2A1 and some hypertrophic chondrocytes COL10A1. In some regions, co-expression of COL1A1 and COL2A1 was observed. At four weeks, such expressions for COL1A1, COL2A1 and COL10A1 became prominent at the area of the fusion mass. From four to six weeks, bone remodelling progressed from the area of the transverse processes towards the central zone. Osteoblasts lining the trabeculae expressed a strong signal for COL1A1. At the central portion of the area of the fusion mass, endochondral ossification progressed and chondrocytes expressed COL2A1 and COL10A1. Our findings show that the fusion process begins with the synthesis of collagens around the transverse processes and around the grafted bone independently. Various spatial and temporal osteogenic and chondrogenic responses, including intramembranous, endochondral and transchondroid bone formation, progress after bone grafting at the intertransverse processes. Bone formation through cartilage may play an important role in posterolateral spinal fusion

The objective of this study was to investigate the effects of different doses rhBMP-2 on bone healing in an ovine lumbar interbody fusion model. In this study 22 sheep underwent two level lumbar interbody fusion using a ventrolateral approach with secondary dorsal fixation at L1/2 and L3/4. After randomization in one level a PEEK-cage was implanted filled with one of three doses rhBMP-2 (0,5mg; 1mg; 2mg) delivered on an ACS. The other level received an empty PEEK-cage or ACS filled cage. Animals were sacrificed after 3 and 6 months and decalcified histology was performed. This included histomorphological analysis well as histomorphometry of the tissues within the cage. At 3 months after surgery the groups treated with rhBMP-2 showed higher amounts of bone tissue within the cage. At 6 months the amounts of bone tissue increased in all groups, were still lower in the groups without growth factor. At 3 months there was only one active osteolysis in the cage/ACS. 7 of 8 segments of the rhBMP-2 groups had a compromised bone structure around the implant. These areas were filled with fibrous tissue and fibrocartilage. This finding was not detected in the groups without rhBMP-2 at 3 months. At 6 months most of the segments with an empty cage or cage/ACS showed a chronic inflammation. Predominant cells were macrophages and giant cells. The groups treated with rhBMP-2 showed only a few mild chronic inflammatory reactions. The well-known dose dependent effect of rhBMP-2 on bone healing could also be recognized in our study. Attention has to be payed to the proinflammatory properties of the growth factor. Consistent with other studies we found 2 strong inflammatory reactions, each one in the lowest and highest dose group. Also, the potential for causing transient bone resorptions, according to the results of others, was demonstrated. At 3 months 7 of 8 segments treated with rhBMP-2 showed compromised peri-implant bone. Osteoblasts, but not osteoclasts, were seen in the periphery of these areas. It can be concluded that there where bone resorptions which already merged into an increased osteoblastic activity. Usually resorptions occur between 2 and 12 weeks and are followed by a period of increased osteoblastic activity. This finding wasn't recognized at 6 months anymore. Striking is that at 6 months most of the segments without rhBMP-2 showed a compromised bone structure around the implant with a mild to mainly moderate chronic inflammatory reaction. This cannot be attributed to the growth factor. Also, the ACS is degraded at 6 months and is unlikely a possible explanation. Therefore, the cage as a reason must be considered and it has to be questioned whether PEEK is the optimal material for interbody cages

Introduction. The objective of this study was to investigate the effects of different doses rhBMP-2 on bone healing in an ovine lumbar interbody fusion model. Methods. In this study 22 sheep underwent two level lumbar interbody fusion using a ventrolateral approach with secondary dorsal fixation at L1/2 and L3/4. After randomization in one level a PEEK-cage was implanted filled with one of three doses rhBMP-2 (0,5mg; 1mg; 2mg) delivered on an ACS. The other level received an empty PEEK-cage or ACS filled cage. Animals were sacrificed after 3 and 6 months and decalcified histology was performed. This included histomorphological analysis as well as histomorphometry of the tissues within the cage. Results. At 3 months after surgery the groups treated with rhBMP-2 showed higher amounts of bone tissue within the cage. At 6 months the amounts of bone tissue increased in all groups, but were still lower in the groups without growth factor. At 3 months there was only one active osteolysis in the cage/ACS. 7 of 8 segments of the rhBMP-2 groups had a compromised bone structure around the implant. These areas were filled with fibrous tissue and fibrocartilage. This finding was not detected in the groups without rhBMP-2 at 3 months. At 6 months most of the segments with an empty cage or cage/ACS showed a chronic inflammation. Predominant cells were macrophages and giant cells. The groups treated with rhBMP-2 showed only a few mild chronic inflammatory reactions. Discussion. The well-known dose dependent effect of rhBMP-2 on bone healing could also be recognized in our study. Attention has to be payed for the proinflammatory properties of the growth factor. Consistent with other studies we found 2 strong inflammatory reactions, each one in the lowest and highest dose group. Also the potential for causing transient bone resorptions, according to the results of others, was demonstrated. At 3 months 7 of 8 segments treated with rhBMP-2 showed compromised peri-implant bone. Osteoblasts, but not osteoclasts, were seen in the periphery of these areas. It can be concluded that there where bone resorptions which already merged into an increased osteoblastic activity. Usually resorptions occur between 2 and 12 weeks and are followed by a period of increased osteoblastic activity. This finding wasn”t recognized at 6 months anymore. Striking is that at 6 months most of the segments without rhBMP-2 showed a compromised bone structure around the implant with a mild to mainly moderate chronic inflammatory reaction. This cannot be attributed to the growth factor. Also the ACS is degraded at 6 months and is unlikely a possible explanation. Therefore, the cage as a reason must be considered and it has to be questioned whether PEEK is the optimal material for interbody cages