Doctor of Philosophy, University Of Helsinki (2013)
Doctor of Medicine, University Of Helsinki (2010)
Stuart Goodman, Postdoctoral Faculty Sponsor
Total joint replacement (TJR) has revolutionized the treatment of end-stage arthritic disorders. This success is due, in large part, to a clear understanding of the important interaction between the artificial implant and the biology of the host. All surgical procedures in which implants are placed in the body evoke an initial inflammatory reaction, which generally subsides over several weeks. Thereafter, a series of homeostatic events occur leading to progressive integration of the implant within bone and the surrounding musculoskeletal tissues. The eventual outcome of the operation is dependent on the characteristics of the implant, the precision of the surgical technique and operative environment, and the biological milieu of the host. If these factors and events are not optimal, adverse events can occur such as the development of chronic inflammation, progressive bone loss due to increased production of degradation products from the implant (periprosthetic osteolysis), implant loosening or infection. These complications can lead to chronic pain and poor function of the joint reconstruction, and may necessitate revision surgery or removal of the prosthesis entirely. Recent advances in engineering, materials science, and the immunological aspects associated with orthopaedic implants have fostered intense research with the hope that joint replacements will last a lifetime, and facilitate pain-free, normal function.
View details for DOI 10.1039/C4TB01005A
View details for PubMedID 25541591
Aseptic loosening of hip replacements is driven by the macrophage reaction to wear particles. The extent of particle-induced macrophage activation is dependent on the state of macrophage polarization, which is dictated by the local cytokine microenvironment. The aim of the study was to characterize cytokine microenvironment surrounding failed, loose hip replacements with an emphasis on identification of cytokines that regulate macrophage polarization. Using qRT-PCR, the expression of interferon gamma (IFN-γ), interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, and IL-17A was low and similar to the expression in control synovial tissues of patients undergoing primary hip replacement. Using immunostaining, no definite source of IFN-γ or IL-4 could be identified. IL-17A positive cells, identified as mast cells by double staining, were detected but their number was significantly reduced in interface tissues compared to the controls. Significant up-regulation of IL-10, M-CSF, IL-8, CCL2-4, CXCL9-10, CCL22, TRAP, cathepsin K, and down regulation of OPG was seen in the interface tissues, while expression of TNF-α, IL-1β, and CD206 were similar between the conditions. It is concluded that at the time of the revision surgery the peri-implant macrophage phenotype has both M1 and M2 characteristics and that the phenotype is regulated by other local and systemic factors than traditional macrophage polarizing cytokines. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
View details for DOI 10.1002/jor.22658
View details for PubMedID 24897980
Aseptic loosening of total joint replacements is driven by the reaction of macrophages to foreign body particles released from the implant. It was hypothesized that the macrophages' response to these particles is dependent, in addition to particle characteristics and contaminating biomolecules, on the state of macrophage polarization as determined by the local cytokine microenvironment. To test this hypothesis we differentiated M1 and M2 macrophages from human peripheral blood monocytes and compared their responses to titanium particles using genome-wide microarray analysis and a multiplex cytokine assay. In comparison to non-activated M0 macrophages, the overall chemotactic and inflammatory responses to titanium particles were greatly enhanced in M1 macrophages and effectively suppressed in M2 macrophages. In addition, the genome-wide approach revealed several novel, potentially osteolytic, particle-induced mediators, and signaling pathway analysis suggested the involvement of toll-like and nod-like receptor signaling in particle recognition. It is concluded that the magnitude of foreign body reaction caused by titanium particles is dependent on the state of macrophage polarization. Thus, by limiting the action of M1 polarizing factors, e.g. bacterial biofilm formation, in peri-implant tissues and promoting M2 macrophage polarization by biomaterial solutions or pharmacologically, it might be possible to restrict wear-particle-induced inflammation and osteolysis.
View details for DOI 10.1016/j.actbio.2013.06.027
View details for Web of Science ID 000326902500044
View details for PubMedID 23827094
Wear particles generated from total joint replacements can stimulate macrophages to release chemokines, such as monocyte chemoattractant protein 1 (MCP-1), which is the most important chemokine regulating systemic and local cell trafficking and infiltration of monocyte/macrophages in chronic inflammation. One possible strategy to curtail the adverse events associated with wear particles is to mitigate migration and activation of monocyte/macrophages. The purpose of this study is to modulate the adverse effects of particulate biomaterials and inflammatory stimuli such as endotoxin by interfering with the biological effects of the chemokine MCP-1. In the current study, the function of MCP-1 was inhibited by the mutant MCP-1 protein called 7ND, which blocks its receptor, the C-C chemokine receptor type 2 (CCR2) on macrophages. Addition of 7ND decreased MCP-1-induced migration of THP-1 cells in cell migration experiments in a dose-dependent manner. Conditioned media from murine macrophages exposed to clinically relevant polymethylmethacrylate (PMMA) particles with/without endotoxin [lipopolysaccharide (LPS)] had a chemotactic effect on human macrophages, which was decreased dramatically by 7ND. 7ND demonstrated no adverse effects on the viability of macrophages, and the capability of mesenchymal stem cells (MSCs) to form bone at the doses tested. Finally, proinflammatory cytokine production was mitigated when macrophages were exposed to PMMA particles with/without LPS in the presence of 7ND. Our studies confirm that the MCP-1 mutant protein 7ND can decrease macrophage migration and inflammatory cytokine release without adverse effects at the doses tested. Local delivery of 7ND at the implant site may provide a therapeutic strategy to diminish particle-associated periprosthetic inflammation and osteolysis. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
View details for DOI 10.1002/jbm.a.34981
View details for PubMedID 24123855
Total joint replacement (TJR) is a very cost-effective surgery for end-stage arthritis. One important goal is to decrease the revision rate especially because TJR has been extended to younger patients. Continuous production of ultra-high molecular weight polyethylene (UHMWPE) wear particles induces macrophage infiltration and chronic inflammation, which can lead to peri-prosthetic osteolysis. Targeting individual pro-inflammatory cytokines directly has not reversed the osteolytic process in clinical trials, due to compensatory upregulation of other pro-inflammatory factors. We hypothesized that targeting the important transcription factor NF-κB could mitigate the inflammatory response to wear particles, potentially diminishing osteolysis. In the current study, we suppressed NF-κB activity in mouse RAW264.7 and human THP1 macrophage cell lines, as well as primary mouse and human macrophages, via competitive binding with double strand decoy oligodeoxynucleotide (ODN) containing an NF-κB binding element. We found that macrophage exposure to UHMWPE particles induced multiple pro-inflammatory cytokine and chemokine expression including TNF-α, MCP1, MIP1α and others. Importantly, the decoy ODN significantly suppressed the induced cytokine and chemokine expression in both murine and human macrophages, and resulted in suppression of macrophage recruitment. The strategic use of decoy NF-κB ODN, delivered locally, could potentially diminish particle-induced peri-prosthetic osteolysis.
View details for DOI 10.1016/j.actbio.2014.04.034
View details for PubMedID 24814879
Biomaterial-induced tissue responses in patients with total joint replacement are associated with the generation of wear particles, which may lead to chronic inflammation and local bone destruction (periprosthetic osteolysis). Inflammatory reactions associated with wear particles are mediated by several important signaling pathways, the most important of which involves the transcription factor NF-κB. NF-κB activation is essential for macrophage recruitment and maturation, as well as the production of pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6 and MCP1. In addition, NF-κB activation contributes to osteoclast differentiation and maturation via RANK/RANKL signaling, which increases bone destruction and reduces bone formation. Targeting individual downstream cytokines directly (such as TNF-α or IL-1β) may not effectively prevent wear particle induced osteolysis. A more logical upstream therapeutic approach may be provided by direct modulation of the core IκB/IKKα/β/NF-κB signaling pathway in the local environment. However, the timing, dose and strategy for administration should be considered. Suppression of chronic inflammation via inhibition of NF-κB activity in patients with malfunctioning joint replacements may be an effective strategy to mitigate wear particle induced periprosthetic osteolysis.
View details for DOI 10.1016/j.actbio.2013.09.034
View details for PubMedID 24090989
Wear particles and by-products from joint replacements and other orthopaedic implants may result in a local chronic inflammatory and foreign body reaction. This may lead to persistent synovitis resulting in joint pain and swelling, periprosthetic osteolysis, implant loosening and pathologic fracture. Strategies to modulate the adverse effects of wear debris may improve the function and longevity of joint replacements and other orthopaedic implants, potentially delaying or avoiding complex revision surgical procedures. Three novel biological strategies to mitigate the chronic inflammatory reaction to orthopaedic wear particles are reported. These include (i) interference with systemic macrophage trafficking to the local implant site, (ii) modulation of macrophages from an M1 (pro-inflammatory) to an M2 (anti-inflammatory, pro-tissue healing) phenotype in the periprosthetic tissues, and (iii) local inhibition of the transcription factor nuclear factor kappa B (NF-κB) by delivery of an NF-κB decoy oligodeoxynucleotide, thereby interfering with the production of pro-inflammatory mediators. These three approaches have been shown to be viable strategies for mitigating the undesirable effects of wear particles in preclinical studies. Targeted local delivery of specific biologics may potentially extend the lifetime of orthopaedic implants.
View details for DOI 10.1098/rsif.2013.0962
View details for PubMedID 24478281
Excessive generation of wear particles after total joint replacement may lead to local inflammation and periprosthetic osteolysis. Modulation of the key transcription factor NF-κB in immune cells could potentially mitigate the osteolytic process. We previously showed that local delivery of ultra-high molecular weight polyethylene (UHMWPE) particles recruited osteoprogenitor cells and reduced osteolysis. However, the biological effects of modulating the NF-κB signaling pathway on osteoprogenitor/mesenchymal stem cells (MSCs) remain unclear. Here we showed that decoy oligodeoxynucleotide (ODN) increased cell viability when primary murine MSCs were exposed to UHMWPE particles, but had no effects on cellular apoptosis. Decoy ODN increased TGF-β1 and osteoprotegerin in MSCs exposed to UHMWPE particles. Mechanistic studies showed that decoy ODN up-regulated osteoprotegerin expression through a TGF-β1 dependent pathway. By measuring alkaline phosphatase activity, osteocalcin levels, Runx2 and osteopontin expression, and performing a bone mineralization assay, we found that decoy ODN increased MSC osteogenic ability when the cells were exposed to UHMWPE particles. Furthermore, the cellular response to decoy ODN and UHMWPE particles with regards to cell phenotype, cell viability and osteogenic ability were confirmed using primary human MSCs. Our results suggest that modulation of wear particle induced inflammation by NF-κB decoy ODN had no adverse effects on MSCs, and may potentially further mitigate peri-prosthetic osteolysis by protecting MSC viability and osteogenic ability.
View details for DOI 10.1089/ten.TEA.2014.0144
View details for PubMedID 25518013
Modulation of macrophage polarization is emerging as promising means to mitigate wear particle-induced inflammation and periprosthetic osteolysis. As a model for continuous local drug delivery, we used miniature osmotic pumps to deliver IL-4 in order to modulate macrophage polarization in vitro from nonactivated M0 and inflammatory M1 phenotypes towards a tissue regenerative M2 phenotype. Pumps delivered IL-4 into vials containing mouse bone marrow macrophage (mBMM) media. This conditioned media (CM) was collected at seven day intervals up to four weeks (week 1 to week 4 samples). IL-4 concentration in the CM was determined by ELISA and its biological activity was assayed by exposing M0 and M1 mBMMs to week 1 or week 4 CM. The IL-4 concentration in the CM approximated the mathematically calculated amount, and its biological activity was well retained, as both M0 and M1 macrophages exposed to either the week 1 or week 4 CM assumed M2-like phenotype as determined by qRT-PCR, ELISA, and immunocytochemistry. The results show that IL-4 can be delivered using osmotic pumps and that IL-4 delivered can modulate macrophage phenotype. Results build a foundation for in vivo studies using our previously validated animal models and provide possible strategies to locally mitigate wear particle-induced macrophage activation and periprosthetic osteolysis. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.
View details for DOI 10.1002/jbm.a.35278
View details for PubMedID 25044942
Total joint replacement (TJR) is a common and effective surgical procedure for hip or knee joint reconstruction. However, the production of wear particles is inevitable for all TJRs, which activates macrophages and initiates an inflammatory cascade often resulting in bone loss, prosthetic loosening and eventual TJR failure. Macrophage Chemoattractant Protein-1 (MCP-1) is one of the most potent cytokines responsible for macrophage cell recruitment, and previous studies suggest that mutant MCP-1 proteins such as 7ND may be used as a decoy drug to block the receptor and reduce inflammatory cell recruitment. Here we report the development of a biodegradable, layer-by-layer (LBL) coating platform that allows efficient loading and controlled release of 7ND proteins from the surface of orthopedic implants using as few as 14 layers. Scanning electron microscopy and fluorescence imaging confirmed effective coating using the LBL procedure on titanium rods. 7ND protein loading concentration and release kinetics can be modulated by varying the polyelectrolytes of choice, the polymer chemistry, the pH of the polyelectrolyte solution, and the degradation rate of the LBL assembly. The released 7ND from LBL coating retained its bioactivity and effectively reduced macrophage migration towards MCP-1. Finally, the LBL coating remained intact following a femoral rod implantation procedure as determined by immunostaining of the 7ND coating. The LBL platform reported herein may be applied for in situ controlled release of 7ND protein from orthopedic implants, to reduce wear particle-induced inflammatory responses in an effort to prolong the lifetime of implants.
View details for DOI 10.1016/j.biomaterials.2013.09.028
View details for Web of Science ID 000328094600054
Degenerating cartilage releases potential danger signals that react with Toll-like receptor (TLR) type danger receptors. We investigated the presence and regulation of TLR1, TLR2, and TLR9 in human chondrocytes.We studied TLR1, TLR2, TLR4, and TLR9 mRNA (qRT-PCR) and receptor proteins (by immunostaining) in primary mature healthy chondrocytes, developing chondrocytes, and degenerated chondrocytes in osteoarthritis (OA) tissue sections of different OARSI grades. Effects of a danger signal and of a pro-inflammatory cytokine on TLRs were also studied.In primary 2D-chondrocytes, TLR1 and TLR2 were strongly expressed. Stimulation of 2D and 3D chondrocytes with a TLR1/2-specific danger signal increased expression of TLR1 mRNA 1.3- to 1.8-fold, TLR2 mRNA 2.6- to 2.8-fold, and TNF-α mRNA 4.5- to 9-fold. On the other hand, TNF-α increased TLR1 mRNA] expression 16-fold, TLR2 mRNA expression 143- to 201-fold, and TNF-α mRNA expression 131- to 265-fold. TLR4 and TLR9 mRNA expression was not upregulated. There was a correlation between worsening of OA and increased TLR immunostaining in the superficial and middle cartilage zones, while chondrocytes assumed a CD166(×) progenitor phenotype. Correspondingly, TLR expression was high soon after differentiation of mesenchymal stem cells to chondrocytes. With maturation, it declined (TLR2, TLR9).Mature chondrocytes express TLR1 and TLR2 and may react to cartilage matrix/chondrocyte-derived danger signals or degradation products. This leads to synthesis of pro-inflammatory cytokines, which stimulate further TLR and cytokine expression, establishing a vicious circle. This suggests that OA can act as an autoinflammatory disease and links the old mechanical wear-and-tear concept with modern biochemical views of OA. These findings suggest that the chondrocyte itself is the earliest and most important inflammatory cell in OA.
View details for DOI 10.3109/17453674.2013.854666
View details for Web of Science ID 000328281100014
View details for PubMedID 24237425
Peri-implant tissue reactions in failed total ankle replacement (TAR) are characterized by early developing peri-implant osteolysis. The hypothesis of the study was that this reaction is mediated by receptor activator of nuclear factor kappa B ligand (RANKL). Samples of peri-prosthetic tissues from failed TAR implants were stained for macrophages, RANKL, its receptor RANK and osteoprotegerin (OPG), and compared to control samples. The failed TAR implants were surrounded by implant capsule, synovial lining-like interface membrane or necrotic tissues. Infiltrating scavenger receptor I positive CD163(+) macrophages were frequent, in particular around necrotic soft tissues or bone sequestrate, and possibly in part formed due to ischemia and mechanical factors. In contrast, implant-derived wear debris was scanty. Still many RANK(+) macrophages were often seen in close contact with RANKL(+) mesenchymal cells, whereas OPG was mostly located at a distance in vascular endothelial cells. Foreign body giant cells were frequent. RANKL seems to stimulate locally accumulated CD163(+) RANK-expressing cells to fusion, which leads to the local formation of multinuclear foreign body giant cells (and probably of osteoclasts). Therefore, peri-implant osteolysis in early TAR implant failure seems to be caused by the RANKL-driven chronic foreign body inflammation directed against, not implant-derived particles, but against necrotic autologous tissues.
View details for DOI 10.1016/j.bone.2012.05.007
View details for Web of Science ID 000307617400029
View details for PubMedID 22627031
Distinction between the two major complications of total hip replacement surgery, septic bacterial culture-positive and aseptic bacterial culture-negative osteolysis and loosening, is difficult due to the eventual role of bacterial remnants and biofilms, which are recognized by cells provided by toll-like receptors (TLRs) of the innate immune system. It was hypothesized that cell typing and TLR mapping might provide new information on the pathomechanisms of loosening. To test this hypothesis, septic (n = 10) and aseptic (n = 5) interface tissue as well as mildly inflamed osteoarthritic synovial membrane (n = 5) samples were characterized and compared using antibodies against several cell line-specific markers, including fibroblast, monocyte/macrophage, T cell, B cell, plasma cell and neutrophil markers, and TLRs. In osteoarthritic synovium, TLR-positive cells were restricted to surface tissues and only few inflammatory cells were detected, whereas aseptic interface was heavily infiltrated with monocyte/macrophages, which were also the major TLR-positive cell type rendering the tissue reactive to TLR ligands. Interestingly, septic cases contained also neutrophil and lymphocyte infiltrates of which especially B-cell infiltrates might be clinically useful in discriminating the two major complications of the joint replacement surgery. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.
View details for DOI 10.1002/jbm.a.32674
View details for PubMedID 20127718
Bacterial remnants and subclinical biofilms residing on prosthesis surfaces have been speculated to play a role in hip implant loosening by opsonizing otherwise relatively inert wear particles. The innate immune system recognizes these microbial pathogen-associated molecular patterns (PAMPs) using Toll-like receptors (TLRs). Our objective was to evaluate the possible presence of TLRs in aseptic synovial membrane-like interface tissue. Bacterial culture-negative, aseptic (n = 4) periprosthetic synovial membrane-like tissue was compared to osteoarthritis synovial membrane (n = 5) for the presence of cells positive for all known human functional TLRs, stained using specific antibodies by immunohistochemistry, and evaluated using morphometry. In comparison to osteoarthtritic synovium, the number of TLR-positive cells was found to be increased in the aseptic setting, reflecting the considerable macrophage infiltration to the tissues investigated. Thus aseptic periprosthetic tissue seems to be very reactive to PAMPs. It has been recently recognized that TLR do not only respond to traditional PAMPs, but also to endogenous alarmings or danger signals released from necrotic and activated cells. Alarming-TLR interaction in the periprosthetic tissue might be a novel mechanism of aseptic loosening of endoprosthesis.
View details for DOI 10.1002/jor.20979
View details for Web of Science ID 000273675700007
View details for PubMedID 19725103
HLA-B27 positive individuals are predisposed to reactive arthritis developing 1-3 weeks after urogenital and gastrointestinal infections. Also ankylosing spondylitis (AS) associates strongly to HLA-B27, but no specific infection, Klebsiella pneumoniae excluded, has been linked to it. Before the discovery of its HLA-B27 association there were many reports suggesting a link between chronic prostatitis in men or pelvic inflammatory disease in women and AS. They have since been forgotten although HLA-B27 did not help to understand, why this disease has an axial and ascending nature. It is proposed that the urogenital organs form a source of damage (or danger)-associated molecular patterns (DAMPs), either exogenous pathogen-associated molecular patterns (PAMPs) from microbes or endogenous alarmins, such as uric acid, released from necrotic cells or urate deposits. DAMPs are slowly seeded from low-down upwards via the pelvic and spinal lymphatic pathways. They reach Toll-like receptors (TLRs) in their target mesenchymal stem cells, which are stimulated to ectopic enchondral bone formation leading to syndesmophytes and bamboo spine. At the same time inflammatory cytokines induce secondary osteoporosis of the spine. This new paradigm places microbes, HLA-B27 and TLRs in the pathogenic centre stage, but without pinpointing any (one) specific pathogen; instead, shared microbial patterns are indicated.
View details for DOI 10.1016/j.jaut.2009.02.010
View details for Web of Science ID 000266510300004
View details for PubMedID 19299108
This study was designed to clarify the role of the receptor activator of nuclear factor kappa B ligand (RANKL) in the process of discus degeneration and spondylarthrosis. It was hypothesized that experimental discus lesion would initiate not only local bone remodelling but also increased osteoclast formation on a location remote to the injury site due to altered spinal biomechanics. It was speculated that these changes in vertebral bone remodelling could be reflected in an increased RANKL expression.The presence of RANKL in the spine was studied in an experimental perforating lesion of the cranial endplate of L4 and the adjoining disc in six domestic pigs and in one human herniated disc. After three months, the experimental and contiguous control vertebrae, complete with intervertebral discs, were subjected for immunohistochemistry.This is the first study to show that RANKL was locally seen (produced) in osteoblasts, fibroblasts replacing annulocytes and mesenchymal bone marrow cells and, in part, apparently bound to the surface of osteoclasts and macrophage-like prefusion macrophages. Such RANKL induction was also seen at sites remote from the experimental lesion driving the whole process. More RANKL-positive cells were found in close proximity to the endplate than in the central parts of the vertebrae. Osteocytes in bone matrix and most bone marrow cells in the marrow microenvironment showed no RANKL staining. Human annulus fibrosus also contained RANKL, RANK and OPG.We have demonstrated that RANKL is produced locally, also in soluble form, at the site of injury and also in intact vertebrae and bony structures likely due to altered biomechanics. It seems to be engaged in bone healing and remodelling, essentially proving our working hypothesis. These secondary bone changes could represent part of the degenerative spine disease (spondylarthrosis). RANKL inhibitors, like recombinant human osteoprotegerin (OPG), could be interesting drugs to test, not only in osteoporosis, but also in spondylarthrosis.
View details for Web of Science ID 000266096700081
View details for PubMedID 19473572