Maria Lennerås

The correlation between gene expression of proinflammatory markers and bone formation during osseointegration with titanium implants.

An in vivo interfacial gene expression model combined with biomechanical analysis was used in order to determine the relationship between the molecular events taking place during osseointegration and the biomechanical stability of the implant. Anodically oxidized and machined, threaded titanium implants were characterized topographically, chemically and ultrastructurally. The implants were inserted in rat tibiae and the implant bone torsion stability was evaluated. After measurements, the implants were removed and analyzed with qPCR. Results showed an increase in the breakpoint torque of 140%, 170% and 190%, after 6, 14, and 28 days, respectively, at the oxidized implants as compared to the machined. Gene expression analysis revealed higher expression of runt related transcription factor-2 (Runx2) (after 28 d), osteocalcin (OC) and tartrate resistant acid phosphatase (TRAP) (after 6, 14 and 28 d) and cathepsin K (CATK) (after 6 and 14 d) at the oxidized implants. On the other hand, machined implants were associated with higher expression of tumor necrosis factor-α (TNF-α) (after 6 and 28 d) and interleukin-1β (IL-1β) (after 6, 14 and 28 d) compared to the oxidized implants. In conclusion, the favorable cellular and molecular events at the oxidized implants were in parallel with significantly stronger bone anchorage during osseointegration.

In vivo gene expression in response to anodically oxidized versus machined titanium implants

A quantitative polymerase chain reaction technique (qPCR) in combination with scanning electron microscopy was applied for the evaluation of early gene expression response and cellular reactions close to titanium implants. Anodically oxidized and machined titanium miniscrews were inserted in rat tibiae. After 1, 3, and 6 days the implants were unscrewed and the surrounding bone was retrieved using trephines. Both the implants and bone were analyzed with qPCR. A greater amount of cells, as indicated with higher expression of 18S, was detected on the oxidized surface after 1 and 6 days. Significantly higher osteocalcin (at day 6), alkaline phosphatase (at days 3 and 6), and cathepsin K (at day 3) expression was demonstrated for the oxidized surface. Higher expression of tumor necrosis factor-alpha (at day 1) and interleukin-1beta (at days 1 and 6) was detected on the machined surfaces. SEM revealed a higher amount of mesenchymal-like cells on the oxidized surface. The results show that the rapid recruitment of mesenchymal cells, the rapid triggering of gene expression crucial for bone remodeling and the transient nature of inflammation, constitute biological mechanisms for osseointegration, and high implant stability associated with anodically oxidized implants.

Free-Form-Fabricated Commercially Pure Ti and Ti6Al4V Porous Scaffolds Support the Growth of Human Embryonic Stem Cell-Derived Mesodermal Progenitors

Commercially-pure titanium (cp-Ti) and the titanium-aluminum-vanadium alloy (Ti6Al4V) are widely used as reconstructive implants for skeletal engineering applications, due to their good mechanical properties, biocompatibility and ability to integrate with the surrounding bone. Electron beam melting technology (EBM) allows the fabrication of customized implants with tailored mechanical properties and high potential in the clinical practice. In order to augment the interaction with the biological tissue, stem cells have recently been combined with metallic scaffolds for skeletal engineering applications. We previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold a great potential to provide a homogeneous and unlimited supply of cells for bone engineering applications. This study demonstrates the effect of EBM-fabricated cp-Ti and Ti6Al4V porous scaffolds on hES-MPs behavior, in terms of cell attachment, growth and osteogenic differentiation. Displaying different chemical composition but similar surface properties, EBM-fabricated cp-Ti and Ti6Al4V scaffolds supported cell attachment and growth, and did not seem to alter the expression of genes involved in osteogenic differentiation and affect the alkaline phosphatase activity. In conclusion, interfacing hES-MPs to EBM-fabricated scaffolds may represent an interesting strategy for design of third-generation biomaterials, with the potential to promote implant integration in clinical conditions characterized by poor bone quality.

Molecular and structural patterns of bone regeneration in surgically created defects containing bone substitutes

Several biomaterials have been introduced for bone augmentation. However, information is lacking about the mechanisms of bone regeneration and/or integration of these materials in the recipient bone. This study aimed to determine the molecular and structural events in bone defects after augmentation with synthetic tetrapod-shaped calcium phosphate (Tetrabone; TetraB) compared with natural deproteinized bovine bone (DBB). Defects were created in the epiphyses of rat femurs and filled with TetraB or DBB or left empty (Sham). After 3, 6, 14 and 28 d, samples were harvested for histology, histomorphometry, ultrastructure and gene expression analyses. At 3 d, higher expressions of bone formation (ALP and OC) and remodeling (CatK) genes were detected in TetraB compared with DBB and Sham. Downregulation of bone remodeling genes (TRAP and CatK) was detected in DBB as compared to Sham after 14 d. Histomorphometry at 6 and 14 d demonstrated greater bone contact with the granules in TetraB. At 28 d, a larger bone area per defect was found in TetraB. The present experiments show that a synthetic substitute, consisting of α-tricalcium and octacalcium phosphates, induces early osteogenic and osteoclastic activities and promotes bone formation in trabecular bone defects.

The influence of bone type on the gene expression in normal bone and at the bone-implant interface: experiments in animal model.

BACKGROUND Studies on the biological processes in different bone types and the reaction of different bone types to biomaterials are often hindered because of the difficulties in sampling procedures and lack of sensitive techniques. PURPOSE The purpose was to assess the suitability of quantitative polymerase chain reaction (qPCR) for investigation of the biological differences between cortical and trabecular bone types and their responses to biomaterials. MATERIALS AND METHODS Gene expression of selected markers in rat bone samples from different locations was evaluated. Samples were harvested by trephines from the trabecular femoral epiphysis, cortico-trabecular proximal tibial metaphysic, and the cortical distal tibial metaphysis. Gene expression was also evaluated at the surfaces of anodically oxidized implants retrieved from cortical and trabecular sites after 3 days of implantation. mRNA in the bone samples and in the tissue associated with the implant surfaces was extracted and quantified using qPCR. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), alkaline phosphatase (ALP), osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK), and 18S ribosomal subunits (18S) were analyzed. RESULTS In the bone samples, higher expression of ALP, OC, TRAP, and CATK was found in femoral epiphysis compared to proximal or distal tibial metaphysis, indicating a higher turnover in the trabecular bone. On the other hand, TNF-α and IL-1β showed higher expression in both tibia sites compared with the femur site, which suggests higher inflammatory potential in the cortical bone. In response to the oxidized implants trabecular bone expressed a higher level of IL-1β, whereas the implants in cortical bone were associated with higher expression of ALP and OC. CONCLUSION There are biological differences between cortical and trabecular bone types, both in the normal steady-state condition and in response to biomaterials. Such differences can be characterized and discriminated quantitatively using a sensitive technique such as qPCR.

Oxidized Titanium Implants Enhance Osseointegration via Mechanisms Involving RANK/RANKL/OPG Regulation.

BACKGROUND The role of implant surface properties for bone formation and bone remodeling, that is, the major events during osseointegration, are incompletely understood. PURPOSE This experimental study aimed to investigate the relation between molecular and morphological patterns at the bone interface for machined and oxidized implants. MATERIALS AND METHODS Machined and anodically oxidized titanium implants were inserted in rat tibiae. The implants and surrounding tissue were retrieved at 1, 3, 6, 14, or 28 days for gene expression, histology, histomorphometry, backscatter scanning electron microscopy, and transmission electron microscopy. RESULTS Compared with machined-surface implants, a higher degree of mineralized bone was found in contact with the oxidized-surface implants. After 3 days, cells adherent to the oxidized implants demonstrated a markedly higher expression of receptor activator of nuclear factor kappa-B (RANK), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG). Whereas the OPG expression was higher at the machined implants at 6, 14, and 28 days, a higher RANKL/OPG ratio was detected at the oxidized implants. Between 3 and 14 days, both implants demonstrated a temporal increase in RANKL/OPG, corresponding to the remodeling phase at the bone-implant interface. For both implant types, the RANKL/OPG ratio sharply decreased to a low level after 28 days. CONCLUSIONS The present results show that oxidized implants rapidly promote a high degree of mineralized bone apposition to the surface. As determined by the gene expression data, the mechanisms involve an early induction of osteoclastic differentiation and subsequently more intensive bone remodeling, which accelerates the maturation of the bone-implant interface. The present study suggests that the RANKL/OPG ratio is a sensitive indicator for monitoring the remodeling process during osseointegration.

Osteogenic response of human mesenchymal stem cells to well-defined nanoscale topography in vitro

Background Patterning medical devices at the nanoscale level enables the manipulation of cell behavior and tissue regeneration, with topographic features recognized as playing a significant role in the osseointegration of implantable devices. Methods In this study, we assessed the ability of titanium-coated hemisphere-like topographic nanostructures of different sizes (approximately 50, 100, and 200 nm) to influence the morphology, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Results We found that the proliferation and osteogenic differentiation of hMSCs was influenced by the size of the underlying structures, suggesting that size variations in topographic features at the nanoscale level, independently of chemistry, can be exploited to control hMSC behavior in a size-dependent fashion. Conclusion Our studies demonstrate that colloidal lithography, in combination with coating technologies, can be exploited to investigate the cell response to well defined nanoscale topography and to develop next-generation surfaces that guide tissue regeneration and promote implant integration.

Locally enhanced early bone formation of zoledronic acid incorporated into a bone cement plug in vivo

The aim of the study was to gain experience about the short‐term effects of zoledronic acid (ZOL) on bone‐implant contact (BIC), bone regeneration and bone area (BA).

Gene Expression of Inflammation and Bone Healing in Peri‐Implant Crevicular Fluid after Placement and Loading of Dental Implants. A Kinetic Clinical Pilot Study Using Quantitative Real‐Time PCR

PURPOSE Early detection of healing complications after placement of dental implants is a pressing but elusive goal. This paper proposes a non-invasive diagnostic tool for monitoring healing- and peri-implant disease specific genes, complementary to clinical evaluations. MATERIAL AND METHODS Eighteen partially edentulous patients were recruited to this pilot study. Three Brånemark TiUnite® implants/patient (Nobel Biocare) were placed in a one-stage procedure. Abutments with smooth or rough (TiUnite®) surface were placed. The test group (n = 9) received fixed bridges (immediate loading), whereas the control group (n = 9) implants were loaded 3 months after surgery. In addition to clinical measurements, crevicular fluid was collected using paper strips at the implant abutments 2, 14, 28, and 90 days postoperative. mRNA was extracted, purified, and converted to cDNA. Quantitative PCR assays for IL-1β, TNF-α, Osteocalcin (OC), Alkaline Phosphatase (ALP), Cathepsin K, Tartrate Resistant Acid Phosphatase, and 18S ribosomal RNA were designed and validated. Relative gene expression levels were calculated. RESULTS One implant was lost in the control group and three in the test group. In one test patient, one implant showed lowered stability after 2 to 4 weeks and was unloaded. Later implant stability improved which allowed for loading after 3 to 4 months. TNF-α and ALP most commonly showed correlation with clinical parameters followed by IL-1β and OC. The strongest correlation was found for TNF-α with clinical complications at 2 and 14 days (p = .01/r = -048, and p = .0004/r = -0.56, respectively; test and control groups together). In some cases, gene expression predicted clinical complications (TNF-α, ALP, CK). CONCLUSION This study is based on samples from few individuals; still, some genes showed correlation with clinical findings. Further studies are needed to refine and optimize the sampling process, to find the appropriate panel, and to validate gene expression for monitoring implant healing.

The clinical, radiological, microbiological, and molecular profile of the skin-penetration site of transfemoral amputees treated with bone-anchored prostheses

Abstract The breach of the skin barrier is a critical issue associated with the treatment of individuals with transfemoral amputation (TFA) using osseointegrated, percutaneous titanium implants. Thirty TFA patients scheduled for abutment exchange or removal were consecutively enrolled. The aims were to determine the macroscopic skin signs, the presence of bacteria and the gene expression in abutment‐adherent cells and to conduct correlative and comparative analyses between the different parameters. Redness and a granulation ring were present in 47% of the patients. Bacteria were detected in 27/30 patients, commonly in the bone canal. Staphylococcus aureus, coagulase‐negative staphylococci, streptococci, and Enterococcus faecalis were the most common. A positive correlation was found between TNF‐α expression and the detection of S. aureus. Staphylococcus aureus together with other bacterial species revealed a positive relationship with MMP‐8 expression. A negative correlation was demonstrated between the length of the residual femur bone and the detection of a granulation ring and E. faecalis. A positive correlation was revealed between fixture loosening and pain and the radiological detection of endosteal bone resorption. Fixture loosening was also correlated with the reduced expression of interleukin‐10 and osteocalcin. It is concluded that several relationships exist between clinical, radiological, microbiological, and molecular assessments of the percutaneous area of TFAs. Further long term studies on larger patient cohorts are required to determine the precise cause‐effect relationships and unravel the role of host‐bacteria interactions in the skin, bone canal and on the abutment for the longevity of percutaneous implants as treatment of TFA.