Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration.

Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/ß isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration.

Periprosthetic UHMWPE Wear Debris Induces Inflammation, Vascularization, and Innervation After Total Disc Replacement in the Lumbar Spine.

BACKGROUND: The pathophysiology and mechanisms driving the generation of unintended pain after total disc replacement (TDR) remain unexplored. Ultrahigh-molecular-weight polyethylene (UHMWPE) wear debris from TDRs is known to induce inflammation, which may result in pain. QUESTIONS/PURPOSES: The purpose of this study was to determine whether (1) periprosthetic UHMWPE wear debris induces immune responses that lead to the production of tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß, the vascularization factors, vascular endothelial growth factor (VEGF) and platelet-derived growth factor-bb (PDGFbb), and the innervation/pain factors, nerve growth factor (NGF) and substance P; (2) the number of macrophages is associated with the production of the aforementioned factors; (3) the wear debris-induced inflammatory pathogenesis involves an increase in vascularization and associated innervation. METHODS: Periprosthetic tissues from our collection of 11 patients with contemporary TDRs were evaluated using polarized light microscopy to quantify UHMWPE wear particles. The major reason for revision (mean implantation time of 3 years [range, 1-6 years]) was pain. For control subjects, biopsy samples from four patients with degenerative disc disease with severe pain and autopsy samples from three normal patients with no history of back pain were also investigated. Immunohistochemistry and histology were used to identify secretory factors, macrophages, and blood vessels. Immunostained serial sections were imaged at ×200 magnification and using MATLAB and NIH ImageJ, a threshold was determined for each factor and used to quantify positive staining normalized to tissue sectional area. The Mann-Whitney U test was used to compare results from different patient groups, whereas the Spearman Rho test was used to determine correlations. Significance was based on p < 0.05. RESULTS: The mean percent area of all six inflammatory, vascularization, and innervation factors was higher in TDR tissues when compared with normal disc tissues. Based on nonparametric data analysis, those factors showing the most significant increase included TNFα (5.17 ± 1.76 versus 0.05 ± 0.03, p = 0.02), VEGF (3.02 ± 1.01 versus 0.02 ± 0.002, p = 0.02), and substance P (4.15 ± 1.01 versus 0.08 ± 0.04, p = 0.02). The mean percent area for IL-1ß (2.41 ± 0.66 versus 0.13 ± 0.13, p = 0.01), VEGF (3.02 ± 1.01 versus 0.34 ± 0.29, p = 0.04), and substance P (4.15 ± 1.01 versus 1.05 ± 0.46, p = 0.01) was also higher in TDR tissues when compared with disc tissues from patients with painful degenerative disc disease. Five of the factors, TNFα, IL-1ß, VEGF, NGF, and substance P, strongly correlated with the number of wear particles, macrophages, and blood vessels. The most notable correlations included TNFα with wear particles (p < 0.001, ρ = 0.63), VEGF with macrophages (p = 0.001, ρ = 0.71), and NGF with blood vessels (p < 0.001, ρ = 0.70). Of particular significance, the expression of PDGFbb, NGF, and substance P was predominantly localized to blood vessels/nerve fibers. CONCLUSIONS: These findings indicate wear debris-induced inflammatory reactions can be linked to enhanced vascularization and associated innervation/pain factor production at periprosthetic sites around TDRs. Elucidating the pathogenesis of inflammatory particle disease will provide information needed to identify potential therapeutic targets and treatment strategies to mitigate pain and potentially avoid revision surgery. LEVEL OF EVIDENCE: Level III, therapeutic study.

Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (µsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with µsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with µsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on µsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd.

Identifying Patient-Specific Pathology in Osteoarthritis Development Based on MicroCT Analysis of Subchondral Trabecular Bone.

The goal of this study was to identify alternative mechanisms of osteoarthritis pathology by analyzing subchondral bone. Femoral condyle samples were collected from post-menopausal female patients with knee osteoarthritis undegoing total knee arthroplasty. In the majority of patients, subchondral trabecular bone volume doubled under a region of the medial femoral condyle with full-thickness cartilage deterioration. However, in a subset of patients the bone volume in this region remained constant. This subset also had larger areas of vascular penetration in the calcified cartilage of the lateral condyle concurrent with increased vascular endothelial growth factor expression. Subtyping by subchondral bone characteristics identified a unique population, which lacked the sclerotic bone characteristic of late-stage osteoarthritis. Identification of subtypes within the osteoarthritis population allows investigation of alternate disease pathologies.

Nonthermal atmospheric pressure plasma enhances mouse limb bud survival, growth, and elongation.

The enhanced differentiation of mesenchymal cells into chondrocytes or osteoblasts is of paramount importance in tissue engineering and regenerative therapies. A newly emerging body of evidence demonstrates that appendage regeneration is dependent on reactive oxygen species (ROS) production and signaling. Thus, we hypothesized that mesenchymal cell stimulation by nonthermal (NT)-plasma, which produces and induces ROS, would (1) promote skeletal cell differentiation and (2) limb autopod development. Stimulation with a single treatment of NT-plasma enhanced survival, growth, and elongation of mouse limb autopods in an in vitro organ culture system. Noticeable changes included enhanced development of digit length and definition of digit separation. These changes were coordinated with enhanced Wnt signaling in the distal apical epidermal ridge (AER) and presumptive joint regions. Autopod development continued to advance for approximately 144 h in culture, seemingly overcoming the negative culture environment usually observed in this in vitro system. Real-time quantitative polymerase chain reaction analysis confirmed the up-regulation of chondrogenic transcripts. Mechanistically, NT-plasma increased the number of ROS positive cells in the dorsal epithelium, mesenchyme, and the distal tip of each phalange behind the AER, determined using dihydrorhodamine. The importance of ROS production/signaling during development was further demonstrated by the stunting of digital outgrowth when anti-oxidants were applied. Results of this study show NT-plasma initiated and amplified ROS intracellular signaling to enhance development of the autopod. Parallels between development and regeneration suggest that the potential use of NT-plasma could extend to both tissue engineering and clinical applications to enhance fracture healing, trauma repair, and bone fusion.

Rare complications of osteolysis and periprosthetic tissue reactions after hybrid and non-hybrid total disc replacement.

PURPOSE: Few complications have been reported for lumbar total disc replacement (TDR) and hybrid TDR fixations. This study evaluated retrieved implants and periprosthetic tissue reactions for two cases of osteolysis following disc arthroplasty with ProDisc-L prostheses. METHODS: Implants were examined for wear and surface damage, and tissues for inflammation, polyethylene wear debris (polarized light microscopy) and metal debris (energy-dispersive X-ray spectroscopy). RESULTS: Despite initial good surgical outcomes, osteolytic cysts were noted in both patients at vertebrae adjacent to the implants. For the hybrid TDR case, heterotopic ossification and tissue necrosis due to wear-induced inflammation were observed. In contrast, the non-hybrid implant showed signs of abrasion and impingement, and inflammation was observed in tissue regions with metal and polyethylene wear debris. CONCLUSIONS: In both cases, wear debris and inflammation may have contributed to osteolysis. Surgeons using ProDisc prostheses should be aware of these rare complications.

UHMWPE wear debris and tissue reactions are reduced for contemporary designs of lumbar total disc replacements.

BACKGROUND: Lumbar total disc replacement (L-TDR) is a procedure used to relieve back pain and maintain mobility. Contemporary metal-on-polyethylene (MoP) L-TDRs were developed to address wear performance concerns about historical designs, but wear debris generation and periprosthetic tissue reactions for these newer implants have not been determined. QUESTIONS/PURPOSES: The purpose of this study was to determine (1) whether periprosthetic ultrahigh-molecular-weight polyethylene (UHMWPE) wear debris and biological responses were present in tissues from revised contemporary MoP L-TDRs that contain conventional cores fabricated from γ-inert-sterilized UHMWPE; (2) how fixed- versus mobile-bearing design affected UHMWPE wear particle number, shape, and size; and (3) how these wear particle characteristics compare with historical MoP L-TDRs that contain cores fabricated from γ-air-sterilized UHMWPE. METHODS: We evaluated periprosthetic tissues from 11 patients who received eight fixed-bearing ProDisc-L and four mobile-bearing CHARITÉ contemporary L-TDRs with a mean implantation time of 4.1 and 2.7 years, respectively. Histologic analysis of tissues was performed to assess biological responses and polarized light microscopy was used to quantify number and size/shape characteristics of UHMWPE wear particles from the fixed- and mobile-bearing devices. Comparisons were made to previously reported particle data for historical L-TDRs. RESULTS: Five of seven (71%) fixed-bearing and one of four mobile-bearing L-TDR patient tissues contained at least 4 particles/mm(2) wear with associated macrophage infiltration. Tissues with wear debris were highly vascularized, whereas those without debris were more necrotic. Given the samples available, the tissue around mobile-bearing L-TDR was observed to contain 87% more, 11% rounder, and 11% less-elongated wear debris compared with tissues around fixed-bearing devices; however, there were no significant differences. Compared with historical L-TDRs, UHMWPE particle number and circularity for contemporary L-TDRs were 99% less (p = 0.003) and 50% rounder (p = 0.003). CONCLUSIONS: In this preliminary study, short-term results suggest there was no significant influence of fixed- or mobile-bearing designs on wear particle characteristics of contemporary L-TDRs, but conventional UHMWPE has notably improved the wear resistance of these devices compared with historical UHMWPE.

Which design and biomaterial factors affect clinical wear performance of total disc replacements? A systematic review.

BACKGROUND: Total disc replacement was clinically introduced to reduce pain and preserve segmental motion of the lumbar and cervical spine. Previous case studies have reported on the wear and adverse local tissue reactions around artificial prostheses, but it is unclear how design and biomaterials affect clinical outcomes. QUESTIONS/PURPOSES: Which design and material factors are associated with differences in clinical wear performance (implant wear and periprosthetic tissue response) of (1) lumbar and (2) cervical total disc replacements? METHODS: We performed a systematic review on the topics of implant wear and periprosthetic tissue response using an advanced search in MEDLINE and Scopus electronic databases. Of the 340 references identified, 33 were retrieved for full-text evaluation, from which 16 papers met the inclusion criteria (12 on lumbar disc replacement and five on cervical disc replacement; one of the included studies reported on both lumbar and cervical disc replacement), which involved semiquantitative analysis of wear and adverse local tissue reactions along with a description of the device used. An additional three papers were located by searching bibliographies of key articles. There were seven case reports, three case series, two case-control studies, and seven analytical studies. The Methodological Index for Non-randomized Studies (MINORS) Scale was used to score case series and case-control studies, which yielded mean scores of 10.3 of 16 and 17.5 of 24, respectively. In general, the case series (three) and case-control (two) studies were of good quality. RESULTS: In lumbar regions, metal-on-polymer devices with mobile-bearing designs consistently generated small and large polymeric wear debris, triggering periprosthetic tissue activation of macrophages and giant cells, respectively. In the cervical regions, metal-on-polymer devices with fixed-bearing designs had similar outcomes. All metal-on-metal constructs tended to generate small metallic wear debris, which typically triggered an adaptive immune response of predominantly activated lymphocytes. There were no retrieval studies on one-piece prostheses. CONCLUSIONS: This review provides evidence that design and biomaterials affect the type of wear and inflammation. However, clinical study design, followup, and analytical techniques differ among investigations, preventing us from drawing firm conclusions about the relationship between implant design and wear performance for both cervical and lumbar total disc replacement.

The role of oxidative stress in aseptic loosening of total hip arthroplasties.

This study investigated the hypothesis that wear particle-induced oxidative stress initiates osteolysis after total hip arthroplasty (THA). Patient radiographs were scored for osteolysis and periprosthetic tissues were immunostained and imaged to quantify polyethylene wear, inflammation, and five osteoinflammatory and oxidative stress-responsive factors. These included high mobility group protein-B1 (HMGB1), cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), 4-hydroxynonenal (4-HNE), and nitrotyrosine (NT). The results show wear debris correlated with inflammation, 4-HNE, NT and HMGB1, whereas inflammation only correlated with NT and HMGB1. Similar to wear debris and inflammation, osteolysis correlated with HMGB1. Additionally, osteolysis correlated with COX2 and 4-HNE, but not iNOS or NT. Understanding the involvement of oxidative stress in wear-induced osteolysis will help identify diagnostic biomarkers and therapeutic targets to prevent osteolysis after THA.

Skeletal cell differentiation is enhanced by atmospheric dielectric barrier discharge plasma treatment.

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing ß-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury.

Retrieval analysis of PEEK rods for posterior fusion and motion preservation.

INTRODUCTION: The purpose of this study was to analyze explanted PEEK rod spinal systems in the context of their clinical indications. We evaluated damage to the implant and histological changes in explanted periprosthetic tissues. METHODS: 12 patients implanted with 23 PEEK rods were revised between 2008 and 2012. PEEK rods were of the same design (CD Horizon Legacy, Medtronic, Memphis TN, USA). Retrieved components were assessed for surface damage mechanisms, including plastic deformation, scratching, burnishing, and fracture. Patient history and indications for PEEK rod implantation were obtained from analysis of the medical records. RESULTS: 11/12 PEEK rod systems were employed for fusion at one level, and motion preservation at the adjacent level. Surgical complications in the PEEK cohort included a small dural tear in one case that was immediately repaired. There were no cases of PEEK rod fracture or pedicle screw fracture. Retrieved PEEK rods exhibited scratching, as well as impressions from the set screws and pedicle screw saddles. PEEK debris was observed in two patient tissues, which were located adjacent to PEEK rods with evidence of scratching and burnishing. CONCLUSION: This study documents the surface changes and tissue reactions for retrieved PEEK rod stabilization systems. Permanent indentations by the set screws and pedicle screws were the most prevalent observations on the surface of explanted PEEK rods.

Severe impingement of lumbar disc replacements increases the functional biological activity of polyethylene wear debris.

BACKGROUND: Wear, oxidation, and particularly rim impingement damage of ultra-high molecular weight polyethylene total disc replacement components have been observed following surgical revision. However, neither in vitro testing nor retrieval-based evidence has shown the effect(s) of impingement on the characteristics of polyethylene wear debris. Thus, we sought to determine (1) differences in polyethylene particle size, shape, number, or biological activity that correspond to mild or severe rim impingement and (2) in an analysis of all total disc replacements, regardless of impingement classification, whether there are correlations between the extent of regional damage and the characteristics of polyethylene wear debris. METHODS: The extent of dome and rim damage was characterized for eleven retrieved polyethylene cores obtained at revision surgery after an average duration of implantation of 9.7 years (range, 4.6 to 16.1 years). Polyethylene wear debris was isolated from periprosthetic tissues with use of nitric acid and was imaged with use of environmental scanning electron microscopy. Subsequently, particle size, shape, number, biological activity, and chronic inflammation scores were determined. RESULTS: Grouping of particles by size ranges that represented high biological relevance (<0.1 to 1-µm particles), intermediate biological relevance (1 to 10-µm particles), and low biological relevance (>10-µm particles) revealed an increased volume fraction of particles in the <0.1 to 1-µm and 1 to 10-µm size ranges in the mild-impingement cohort as compared with the severe-impingement cohort. The increased volume fractions resulted in a higher specific biological activity per unit particle volume in the mild-impingement cohort than in the severe-impingement cohort. However, functional biological activity, which is normalized by particle volume (mm3/g of tissue), was significantly higher in the severe-impingement cohort. This increase was due to a larger volume of particles in all three size ranges. In both cohorts, the functional biological activity correlated with the chronic inflammatory response, and the extent of rim penetration positively correlated with increasing particle size, number, and functional biological activity. CONCLUSIONS: The results of this study suggest that severe rim impingement increases the production of biologically relevant particles from motion-preserving lumbar total disc replacement components. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Characterization of wear debris in total elbow arthroplasty.

BACKGROUND: The purpose of this study was to evaluate wear debris in periprosthetic tissues at the time of revision total elbow arthroplasty. Polyethylene, metallic, and bone cement debris were characterized, and the tissue response was quantified. MATERIALS AND METHODS: Capsular and medullary tissue samples were collected during revision surgery. Polyethylene debris was characterized by scanning electron microscopy after tissue digestion. The concentrations of metal and cement debris were quantified by inductively coupled plasma mass spectrometry. Tissue response was graded with a semiquantitative histologic method. RESULTS: Polyethylene particle size varied from the submicron range to over 100 µm. The mean diameter ranged from 0.6 µm to about 1 µm. Particles in the synovial tissues were larger and less abundant than those in tissues from the medullary canal. Cement, titanium alloy, and low levels of cobalt-chrome debris were also present, with cement predominating over metal debris. Histiocyte response was associated with small polyethylene particles (0.5-2 µm), and giant cells were associated with large polyethylene particles (>2 µm). Histiocyte scores positively correlated with the polyethylene particle number and the presence of metal. DISCUSSION: We have shown that periprosthetic tissues of total elbow patients who have undergone revision for loosening and osteolysis contain polyethylene, cement, and metal debris. Although the polyethylene particles were of a size and shape that have been previously shown to result in activation of phagocytic cells, osteolysis after total elbow arthroplasty is a multimodal process. Because of the presence of multiple wear particle sources, a cause-and-effect relationship between polyethylene debris and osteolysis cannot be established with certainty.

Characteristics of highly cross-linked polyethylene wear debris in vivo.

Despite the widespread implementation of highly cross-linked polyethylene (HXLPE) liners to reduce the clinical incidence of osteolysis, it is not known if the improved wear resistance will outweigh the inflammatory potential of HXLPE wear debris generated in vivo. Thus, we asked: What are the differences in size, shape, number, and biological activity of polyethylene wear particles obtained from primary total hip arthroplasty revision surgery of conventional polyethylene (CPE) versus remelted or annealed HXLPE liners? Pseudocapsular tissue samples were collected from revision surgery of CPE and HXLPE (annealed and remelted) liners, and digested using nitric acid. The isolated polyethylene wear particles were evaluated using scanning electron microscopy. Tissues from both HXLPE cohorts contained an increased percentage of submicron particles compared to the CPE cohort. However, the total number of particles was lower for both HXLPE cohorts, as a result there was no significant difference in the volume fraction distribution and specific biological activity (SBA; the relative biological activity per unit volume) between cohorts. In contrast, based on the decreased size and number of HXLPE wear debris there was a significant decrease in total particle volume (mm(3)/g of tissue). Accordingly, when the SBA was normalized by total particle volume (mm(3)/gm tissue) or by component wear volume rate (mm(3)/year), functional biological activity of the HXLPE wear debris was significantly decreased compared to the CPE cohort. Indications for this study are that the osteolytic potential of wear debris generated by HXLPE liners in vivo is significantly reduced by improvements in polyethylene wear resistance.

Do tissues from THA revision of highly crosslinked UHMWPE liners contain wear debris and associated inflammation?

BACKGROUND: Polyethylene wear debris is a major contributor to inflammation and the development of implant loosening, a leading cause of THA revisions. To reduce wear debris, highly crosslinked ultrahigh-molecular-weight polyethylene (UHMWPE) was introduced to improve wear properties of bearing surfaces. As highly crosslinked UHMWPE revision tissues are only now becoming available, it is possible to examine the presence and association of wear debris with inflammation in early implant loosening. QUESTIONS/PURPOSES: We asked: (1) Does the presence of UHMWPE wear debris in THA revision tissues correlate with innate and/or adaptive immune cell numbers? (2) Does the immune cell response differ between conventional and highly crosslinked UHMWPE cohorts? METHODS: We collected tissue samples from revision surgery of nine conventional and nine highly crosslinked UHMWPE liners. Polarized light microscopy was used to determine 0.5- to 2-µm UHMWPE particle number/mm2, and immunohistochemistry was performed to determine macrophage, T cell, and neutrophil number/mm2. RESULTS: For the conventional cohort, correlations were observed between wear debris and the magnitude of individual patient macrophage (ρ=0.70) and T cell responses (ρ=0.71) and between numbers of macrophages and T cells (ρ=0.77) in periprosthetic tissues. In comparison, the highly crosslinked UHMWPE cohort showed a correlation between wear debris and the magnitude of macrophage responses (ρ=0.57) and between macrophage and T cell numbers (ρ=0.68). Although macrophages and T cells were present in both cohorts, the highly crosslinked UHMWPE cohort had lower numbers, which may be associated with shorter implantation times. CONCLUSIONS: The presence of wear debris and inflammation in highly crosslinked UHMWPE revision tissues may contribute to early implant loosening.

Mast cells and hypoxia drive tissue metaplasia and heterotopic ossification in idiopathic arthrofibrosis after total knee arthroplasty.

BACKGROUND: Idiopathic arthrofibrosis occurs in 3-4% of patients who undergo total knee arthroplasty (TKA). However, little is known about the cellular or molecular changes involved in the onset or progression of this condition. To classify the histomorphologic changes and evaluate potential contributing factors, periarticular tissues from the knees of patients with arthrofibrosis were analyzed for fibroblast and mast cell proliferation, heterotopic ossification, cellular apoptosis, hypoxia and oxidative stress. RESULTS: The arthrofibrotic tissue was composed of dense fibroblastic regions, with limited vascularity along the outer edges. Within the fibrotic regions, elevated numbers of chymase/fibroblast growth factor (FGF)-expressing mast cells were observed. In addition, this region contained fibrocartilage and associated heterotopic ossification, which quantitatively correlated with decreased range of motion (stiffness). Fibrotic, fibrocartilage and ossified regions contained few terminal dUTP nick end labeling (TUNEL)-positive or apoptotic cells, despite positive immunostaining for lactate dehydrogenase (LDH)5, a marker of hypoxia, and nitrotyrosine, a marker for protein nitrosylation. LDH5 and nitrotyrosine were found in the same tissue areas, indicating that hypoxic areas within the tissue were associated with increased production of reactive oxygen and nitrogen species. CONCLUSIONS: Taken together, we suggest that hypoxia-associated oxidative stress initiates mast cell proliferation and FGF secretion, spurring fibroblast proliferation and tissue fibrosis. Fibroblasts within this hypoxic environment undergo metaplastic transformation to fibrocartilage, followed by heterotopic ossification, resulting in increased joint stiffness. Thus, hypoxia and associated oxidative stress are potential therapeutic targets for fibrosis and metaplastic progression of idiopathic arthrofibrosis after TKA.

Distinct immunohistomorphologic changes in periprosthetic hip tissues from historical and highly crosslinked UHMWPE implant retrievals.

Assessment of immune response to implant wear debris in periprosthetic tissue following total hip arthroplasty suggests that multiple factors are involved in the loss implant function. The current study investigated wear debris and the associated immunohistomorphologic changes in tissues from nine patients with historical (gamma air-sterilized) and nine highly crosslinked UHMWPE implant components. Paraffin embedded tissue sections were evaluated for the presence of histiocytes, giant cells, fibrocartilage/bone, and necrosis. To determine the incidence, degree and co-localization of immunohistomorphologic changes and wear, overlapping full-field tissue arrays were collected in brightfield and polarized light. The historical cohort tissues predominantly showed histiocytes associated with significant accumulations of small wear (0.5-2 microm), and giant cells associated with large wear (> or =2 microm). Frequently, focal regions of necrosis were observed in association with wear debris. For the highly crosslinked cohort, inflammation and associated wear debris were limited, but in tissues from patients revised after implantation times of >2 years a response was observed. Whereas significant amounts of fibrocartilage/bone were observed in patients at earlier implantation times. In both cohorts, tissue responses were more extensive in the retroacetabular or proximal femoral regions. The current findings suggest that wear debris-induced inflammation may be a major contributor to the loss of implant function for both the historical and highly crosslinked cohorts, but it is not the primary cause of early implant loosening. This study highlights the importance of using a more quantitative and standardized assessment of immunohistomorphologic responses in periprosthetic tissues, and emphasizes differences in specific anatomical regions of individual patient tissues.

Reactive oxygen and nitrogen species induce protein and DNA modifications driving arthrofibrosis following total knee arthroplasty.

BACKGROUND: Arthrofibrosis, occurring in 3%-4% of patients following total knee arthroplasty (TKA), is a challenging condition for which there is no defined cause. The hypothesis for this study was that disregulated production of reactive oxygen species (ROS) and nitrogen species (RNS) mediates matrix protein and DNA modifications, which result in excessive fibroblastic proliferation. RESULTS: We found increased numbers of macrophages and lymphocytes, along with elevated amounts of myeloperoxidase (MPO) in arthrofibrotic tissues when compared to control tissues. MPO expression, an enzyme that generates ROS/RNS, is usually limited to neutrophils and some macrophages, but was found by immunohistochemistry to be expressed in both macrophages and fibroblasts in arthrofibrotic tissue. As direct measurement of ROS/RNS is not feasible, products including DNA hydroxylation (8-OHdG), and protein nitrosylation (nitrotyrosine) were measured by immunohistochemistry. Quantification of the staining showed that 8-OHdg was significantly increased in arthrofibrotic tissue. There was also a direct correlation between the intensity of inflammation and ROS/RNS to the amount of heterotopic ossification (HO). In order to investigate the aberrant expression of MPO, a real-time oxidative stress polymerase chain reaction array was performed on fibroblasts isolated from arthrofibrotic and control tissues. The results of this array confirmed the upregulation of MPO expression in arthrofibrotic fibroblasts and highlighted the downregulated expression of the antioxidants, superoxide dismutase1 and microsomal glutathione S-transferase 3, as well as the significant increase in thioredoxin reductase, a known promoter of cell proliferation, and polynucleotide kinase 3'-phosphatase, a key enzyme in the base excision repair pathway for oxidative DNA damage. CONCLUSION: Based on our current findings, we suggest that ROS/RNS initiate and sustain the arthrofibrotic response driving aggressive fibroblast proliferation and subsequent HO.

Comparison of periprosthetic tissue digestion methods for ultra-high molecular weight polyethylene wear debris extraction.

There is considerable interest in characterization of wear debris from polyethylene (UHMWPE) bearing components used in total joint replacement. To isolate UHMWPE wear debris, tissue samples must be excised from regions adjacent to revised UHMWPE implant components, followed by exposure to one of many available tissue digestion methods. Numerous studies demonstrate successful digestion, but the relative efficiency of each method is not clear. The purpose of this study was to evaluate a variety of conditions for tissue digestion to provide a quantitative comparison of methods. Porcine and human hip tissues were exposed for 24 h to basic, acidic or enzymatic agents, filtered and digestion efficiency calculated based on the percentage of initial to final tissue weight. Of the conditions tested, 5 M NaOH, 5 M KOH, 15 M KOH or 15.8 M HNO(3) yielded the most complete porcine hip tissue digestion (<1% residual tissue weight; p < 0.05). Proteinase K and Liberase Blendzyme 3 did not effectively digest tissue in a 24 h period. Similar to results from the porcine dataset, human tissues digestion was most efficient using 5 M NaOH, 5 M KOH or 15.8 M HNO(3) (<1% residual tissue weight; p < 0.05). To verify that particle surface modifications did not occur after prolonged reagent exposure, GUR415 and Ceridust 3715 particles were immersed in each solution for 24 h. Overall, this study provides a framework for thorough and efficient digestive methods for UHMWPE wear debris extraction.

Retrieval analysis of a ProDisc-L total disc replacement.

STUDY DESIGN: We retrieved a functioning ProDisc-L total disc replacement and associated tissues at 16 months of service life. OBJECTIVE: To analyze a previously unreported mode of implant malpositioning, wear mechanisms, and polyethylene locking mechanism, and to study retrieved periprosthetic tissues. SUMMARY OF BACKGROUND DATA: The clinical performance of polyethylene in the context of total disc replacements remains poorly understood. In the ProDisc-L, the polyethylene core is fixed to the inferior metal endplate through a mechanical interference locking mechanism similar to those used in tibial total knee components. This case represents the third report of an explanted ProDisc-L prosthesis, and the first reported case of posterior malpositioning with this device. METHODS: The implant was removed via a transperitoneal approach. Its polyethylene core was evaluated for burnishing, fracture, third-body abrasion, and permanent deformation. An identical, never-implanted set of polyethylene and endplate components served as controls for the microscopic evaluation of wear. Two tissue samples were collected from a region adjacent to the failed implant to evaluate tissue morphology and inflammation. Hematoxylin and eosin-stained tissue sections were also evaluated for the presence of polyethylene debris by polarized light microscopy. RESULTS: The implant was removed without serious incident, although there were incidental venotomies. The patient went on to solid arthrodesis. We found minimal wear, oxidation, and periprosthetic tissue reaction, as might be expected given the short-term duration of implantation and its reason for revision. No evidence was found of malfunction or improper deployment of the locking mechanism. Burnishing seemed to be the result of short-term impingement. Some areas of the tissue matrix showed evidence of early cell degeneration, and some of these areas contained polyethylene particles identified by polarized light microscopy. CONCLUSIONS: A larger series of implant retrievals will be needed to investigate possible wear and the biologic response to increased particle generation.