Jukka Pajarinen

Inflammation, fracture and bone repair

The reconstitution of lost bone is a subject that is germane to many orthopedic conditions including fractures and non-unions, infection, inflammatory arthritis, osteoporosis, osteonecrosis, metabolic bone disease, tumors, and periprosthetic particle-associated osteolysis. In this regard, the processes of acute and chronic inflammation play an integral role. Acute inflammation is initiated by endogenous or exogenous adverse stimuli, and can become chronic in nature if not resolved by normal homeostatic mechanisms. Dysregulated inflammation leads to increased bone resorption and suppressed bone formation. Crosstalk among inflammatory cells (polymorphonuclear leukocytes and cells of the monocyte-macrophage-osteoclast lineage) and cells related to bone healing (cells of the mesenchymal stem cell-osteoblast lineage and vascular lineage) is essential to the formation, repair and remodeling of bone. In this review, the authors provide a comprehensive summary of the literature related to inflammation and bone repair. Special emphasis is placed on the underlying cellular and molecular mechanisms, and potential interventions that can favorably modulate the outcome of clinical conditions that involve bone repair.

Macrophages – Key Cells in the Response to Wear Debris from Joint Replacements

The generation of wear debris is an inevitable result of normal usage of joint replacements. Wear debris particles stimulate local and systemic biological reactions resulting in chronic inflammation, periprosthetic bone destruction, and eventually, implant loosening, and revision surgery. The latter may be indicated in up to 15% patients in the decade following the arthroplasty using conventional polyethylene. Macrophages play multiple roles in both inflammation and in maintaining tissue homeostasis. As sentinels of the innate immune system, they are central to the initiation of this inflammatory cascade, characterized by the release of proinflammatory and pro-osteoclastic factors. Similar to the response to pathogens, wear particles elicit a macrophage response, based on the unique properties of the cells belonging to this lineage, including sensing, chemotaxis, phagocytosis, and adaptive stimulation. The biological processes involved are complex, redundant, both local and systemic, and highly adaptive. Cells of the monocyte/macrophage lineage are implicated in this phenomenon, ultimately resulting in differentiation and activation of bone resorbing osteoclasts. Simultaneously, other distinct macrophage populations inhibit inflammation and protect the bone-implant interface from osteolysis. Here, the current knowledge about the physiology of monocyte/macrophage lineage cells is reviewed. In addition, the pattern and consequences of their interaction with wear debris and the recent developments in this field are presented.

Chronic inflammation in biomaterial-induced periprosthetic osteolysis: NF-κB as a therapeutic target

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.

Novel biological strategies for treatment of wear particle-induced periprosthetic osteolysis of orthopaedic implants for joint replacement

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.

The effects of immunomodulation by macrophage subsets on osteogenesis in vitro

BackgroundBone formation and remodeling are influenced by the inflammatory state of the local microenvironment. In this regard, macrophages are postulated to play a crucial role in modulating osteogenesis. However, the differential effects of macrophage subsets and their plasticity on bone formation are currently unknown.MethodsPolarized primary murine macrophages and preosteoblastic MC3T3 cells were co-cultured to investigate the effect of non-activated M0, pro-inflammatory M1, and tissue-regenerative M2 macrophages on the osteogenic ability of MC3T3-E1 cells in vitro. Furthermore, to model the physiological transition from inflammation to tissue regeneration, M1-MC3T3 co-cultures were treated with interleukin-4 (IL-4) at different time points to modulate the M1 phenotype towards M2. Macrophage phenotypic markers were assessed by flow cytometry and enzyme-linked immunosorbent assay. A time course study of osteogenic markers at different time points was conducted: alkaline phosphatase (ALP) mRNA levels were evaluated at week 1, ALP activity and osteocalcin and osteopontin mRNA levels at week 2, and matrix mineralization and osteocalcin and osteopontin protein concentrations at week 3. Supernatant collected 72 hours after seeding or IL-4 treatment, whichever was later, was analyzed for oncostatin M, a cytokine released by macrophages that has been recognized to enhance osteogenesis. Unpaired t test or one-way ANOVA with Tukey’s or Dunnett’s post hoc tests were used for statistical comparison of the groups.ResultsCo-culture with any of the macrophage subtypes increased the osteogenic ability of MC3T3 cells as indicated by increases in ALP activity and matrix mineralization. Increased ALP activity, osteocalcin concentration, and matrix mineralization demonstrated that osteogenesis by M1-MC3T3 co-cultures was further enhanced by macrophage phenotype modulation to M2 via IL-4 treatment 72 hours after seeding. Increased oncostatin M protein concentration in untreated M1-MC3T3 co-cultures and M1-MC3T3 co-cultures treated with IL-4 at 72 hours correlated with greater ALP activity and matrix mineralization.ConclusionsThese results suggest that a transient inflammatory phase is crucial for enhanced bone formation. Macrophage plasticity may offer new strategies for modulating the local inflammatory microenvironment with the aim of potentially enhancing bone repair.

Toll-like receptors in human chondrocytes and osteoarthritic cartilage.

Background and purpose 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. Methods 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. Results 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). Interpretation 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.

Toll-like receptors and aseptic loosening of hip endoprosthesis—a potential to respond against danger signals?

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.

Mutant MCP-1 protein delivery from layer-by-layer coatings on orthopedic implants to modulate inflammatory response.

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.

Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants

Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure.

Characterization of macrophage polarizing cytokines in the aseptic loosening of total hip replacements

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.