Paul A. Manner

Transforming Growth Factor-β-mediated Chondrogenesis of Human Mesenchymal Progenitor Cells Involves N-cadherin and Mitogen-activated Protein Kinase and Wnt Signaling Cross-talk

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-β (TGF-β) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-βs in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-β1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-β1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-β1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular β-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.

Characterization of Multipotential Mesenchymal Progenitor Cells Derived from Human Trabecular Bone

The in vitro culture of human trabecular bone‐derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase‐treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone‐derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73+, STRO‐1+, CD105+, CD34−, CD45−, CD144− cell population resident within collagenase‐treated, culture‐processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long‐term culture expansion. When cultured as described, the trabecular bone‐derived cells display stem cell‐like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long‐term in vitro culture.

Direct and indirect induction of apoptosis in human mesenchymal stem cells in response to titanium particles.

The most frequent complication of total joint arthroplasty is periprosthetic osteolysis initiated by an inflammatory response to orthopaedic wear debris, which if left untreated, can result in implant instability and failure, eventually requiring revision surgery. We have previously reported that osteogenic differentiation of human marrow stroma‐derived mesenchymal stem cells (hMSCs) is suppressed upon exposure to titanium particles, accompanied by reduced bone sialoprotein (BSP) gene expression, diminished production of collagen type I and BSP, decreased cellular viability and proliferation, and inhibition of extracellular matrix mineralization. In this study, we have further investigated hMSC cytotoxicity upon exposure to submicron particles of commercially pure titanium (cpTi) and zirconium oxide (ZrO2). Our results showed that direct exposure to cpTi and ZrO2 particles compromises cell viability through the induction of apoptosis, eliciting increased levels of the tumor suppressor proteins p53 and p73, in a manner dependent on material composition, particle dosage, and time. Additionally, conditioned medium collected from hMSCs exposed to cpTi particles, but not to ZrO2 particles, is cytotoxic to hMSCs, inducing apoptosis in the absence of particles. These findings demonstrate that exposure to orthopaedically derived wear particles can compromise hMSC viability through the direct and indirect induction of apoptosis. Thus, prolonged in vivo exposure of marrow‐derived hMSCs to implant‐derived wear debris is likely to reduce the population of viable osteoprogenitor cells, and may contribute to poor periprosthetic bone quality and implant loosening.

The American Academy of Orthopaedic Surgeons evidence-based guideline on: treatment of osteoarthritis of the knee, 2nd edition.

The AAOS Evidence-Based Guideline on Treatment of Osteoarthritis of the Knee, 2nd Edition, includes only less-invasive alternatives to knee replacement. This brief summary of the AAOS Clinical Practice Guideline contains a list of the recommendations and the rating of strength based on the quality of the supporting evidence. Discussion of how each recommendation was developed and the complete evidence report are contained in the full guideline at www.aaos.org/guidelines. ### Conservative Treatments: Recommendations 1-6 #### RECOMMENDATION 1 We recommend that patients with symptomatic osteoarthritis of the knee participate in self-management programs, strengthening, low-impact aerobic exercises, and neuromuscular education; and engage in physical activity consistent with national guidelines. Strength of Recommendation: Strong #### RECOMMENDATION 2 We suggest weight loss for patients with symptomatic osteoarthritis of the knee and a BMI ≥25. Strength of Recommendation: Moderate #### RECOMMENDATION 3A We cannot recommend using acupuncture in patients with symptomatic osteoarthritis of …

Comparison of one and two-stage revision of total hip arthroplasty complicated by infection: a Markov expected-utility decision analysis.

BACKGROUND Two-stage revisions of total hip arthroplasties complicated by chronic infection result in reinfection rates that are lower than those following single-stage revisions but may also result in increased surgical morbidity. Using a decision analysis, we compared single-stage and two-stage revisions to determine which treatment modality resulted in greater quality-adjusted life years (QALYs). METHODS A review of the literature on the treatment of patients with an infection at the site of a total hip arthroplasty provided probabilities; utility values for common postoperative health states were determined in a previously published study. With these data, we conducted a Markov cohort simulation decision analysis. Sensitivity analysis validated the model, and comparisons were made in terms of QALYs. RESULTS The twelve-month model favored direct-exchange revision over the two-stage approach, regardless of whether surgeon or patient-derived utilities were used (0.945 versus 0.896 and 0.897 versus 0.861 QALYs for the patient and surgeon models, respectively). Similar results were observed in a lifetime model with a ten-year life expectancy (7.853 versus 7.771, and 7.438 versus 7.362 QALYs, respectively). The findings were found to be robust in sensitivity analyses in which clinically relevant ranges of input variables were used. CONCLUSIONS This analysis favored the direct-exchange arthroplasty over the two-stage approach. This study should be considered hypothesis-generating for future randomized controlled trials in which, ideally, health end points will be considered in addition to the eradication of infection.

Simulated Joint Infection Assessment by Rapid Detection of Live Bacteria with Real-Time Reverse Transcription Polymerase Chain Reaction

BACKGROUND Although microbiological bacterial culture is currently considered the gold standard for diagnosis of septic arthritis, many studies have documented substantial false-negative and false-positive rates. The objective of this study was to determine whether real-time quantitative reverse transcription polymerase chain reaction can be used to detect bacterial messenger RNA (mRNA) in synovial fluid as a way to distinguish live and dead bacteria as an indicator of active infection. METHODS Synovial fluid samples were obtained from twelve consecutive patients who presented with knee pain and effusion but no evidence of infection. Following assurance of sterility with plate cultures, each sample was inoculated with clinically relevant bacteria and incubated for twenty-four hours to simulate septic arthritis. Bacterial viability and load were assessed with cultures. Selected samples were also treated with a single dose of a combination of two antibiotics, vancomycin and gentamicin, and sampled at several time points. Total RNA isolated from each sample was analyzed in triplicate with one-step real-time quantitative reverse transcription polymerase chain reaction to detect mRNA encoding for the genes groEL or femC. Controls included sterile, uninoculated samples and inoculated samples analyzed with quantitative polymerase chain reaction without reverse transcription. mRNA content was estimated on the basis of detection limits as a function of serial dilutions and was expressed as a function of colony number in bacterial cultures and RNA content as determined spectrophotometrically. RESULTS All synovial fluid samples that had been inoculated with one of the four bacterial species, and analyzed in triplicate, were identified (distinguished from aseptic synovial fluid) with real-time quantitative reverse transcription polymerase chain reaction; there were no false-negative results. All inoculated samples produced bacterial colonies on culture plates, while cultures of the aseptic samples were negative for growth. The detection limit of the one-step bacterial mRNA-based real-time quantitative reverse transcription polymerase chain reaction varied depending on the bacterial species. A time-dependent decrease in the concentration of detectable bacterial mRNA was seen after incubation of bacteria with antibiotics. CONCLUSIONS The direct quantification of the concentration of viable bacterial mRNA with real-time quantitative reverse transcription polymerase chain reaction allows identification of both culture-positive bacterial infection and so-called unculturable bacterial infection in a simulated septic arthritis model. In contrast to conventional polymerase chain reaction, real-time quantitative reverse transcription polymerase chain reaction minimizes false-positive detection of nonviable bacteria and thus provides relevant information on the success or failure of antibiotic therapy.

Modern metal-on-metal hip implants

This Technology Overview was prepared using systematic review methodology and summarizes the findings of studies published as of July 15, 2011, on modern metal-on-metal hip implants. Analyses conducted on outcomes by two joint registries indicate that patients who receive metal-on-metal total hip arthroplasty (THA) and hip resurfacing are at greater risk for revision than are patients who receive THA using a different bearing surface combination. Data from these registries also indicate that larger femoral head components have higher revision rates and risk of revision and that older age is associated with increased revision risks of large-head metal-on-metal THA. Several studies noted a correlation between suboptimal hip implant positioning and higher wear rates, local metal debris release, and consequent local tissue reactions to metal debris. In addition, several studies reported elevated serum metal ion concentrations in patients with metal-on-metal hip articulations, although the clinical significance of these elevated ion concentrations remains unknown.

Expert Opinion and Controversies in Musculoskeletal and Sports Medicine: Femoroacetabular Impingement

Femoroacetabular impingement (FAI) has been proposed as a distinct clinical entity that may be a potentially significant cause of hip osteoarthritis (OA). There is a growing body of literature on this condition, including descriptions of biomechanic mechanisms of impingement, anatomic and radiographic findings, and surgical interventions. Although a connection between anatomic abnormalities of the hip and the development of OA has been recognized for some time, there are limited data on the natural history of FAI and no long-term studies on the effect of surgical treatment. Thus, the diagnosis engenders a degree of controversy in multiple regards, including the diagnostic criteria and the role of operative intervention.

Editorial: Fairness to all: Gender and sex in scientific reporting

Women do not benefit from medical research as much as men do [2, 5]. This problem stems both from research design (which scientists largely control), and from scientific reporting in journals (which editors can influence). Starting with the latter, we first must learn to talk about sex. Sex (female or male) refers to the genotype, while gender (woman or girl, man or boy) refers to the social constructs that overlie the genotype. Gender tends to be culturally laden, and as such, separating sex from gender in certain kinds of research is nearly impossible. Do women as a cultural norm have less pain after knee replacement, or is it a condition innate to the biology of the female sex? If the former, might this be driven by women reporting pain differently on standardized scoring instruments (or to their surgeons, who are more likely to be men)? Or are there important physiological differences in pain signaling between males and females? Other explanations are possible — for example, it is likely that women’s responses are interpreted differently by surgeons of either gender — and it is not always possible to know whether sex, gender, or both account for the observed effect. But when possible, we will seek clarity in authors’ explanations: Are the differences gender-driven, sex-driven, or is it not possible to tell? This is not just a semantic issue. It is a health issue, both for women and men. Women have been underrepresented in medical research, and therefore the evidence that drives their care is less robust [2, 5]. Pharmacokinetics and responses to important therapeutic interventions differ between men and women [3], and women are more likely to experience adverse drug reactions [6]. Surgeons may believe this is a “medical thing” and not a problem in orthopaedic surgery. That is wrong. Women consume approximately 85% of the Cox-II-specific NSAIDs that are prescribed and the side effect profiles of these drugs — including important, life-threatening side effects — differ between men and women [6]. Yet, the treatment of women with NSAIDs is based on Cox-II trials consisting disproportionately of men [1]. We learned only belatedly that women are at much greater risk of complications and failure after total hip resurfacing arthroplasty [4], and the result suggests that clearer scientific reporting would have prevented harm to many women. We probably do not know the full extent of the harm we may be causing because the reporting of results by gender is so inconsistently performed in medical and surgical trials in our specialty. This must change. Accordingly, we recommend that investigators writing for CORR®: Design studies that are sufficiently powered to answer research questions both for males and females (or men and women) if the health condition being studied occurs in both sexes/genders. Provide sex- and/or gender-specific data where relevant in all clinical, basic science, and epidemiological studies. Analyze the influence (or association) of sex or gender on the results of the study, or indicate in the Patients and Methods section why such analyses were not performed, and consider this topic as a limitation to cover in the Discussion section. Readers need to know whether the results generalize to both sexes/genders. Indicate (if sex or gender analyses were performed post-hoc) that these analyses should be interpreted cautiously because they may be underpowered (leading to a false conclusion of no difference). If there are many such analyses, indicate that they may lead to spurious significance, and an erroneous conclusion of a sex- or gender-related difference. We present these as recommendations, rather than requirements for publication because the topic is relatively new to the collective consciousness of our specialty. Our editorial board will continue to evaluate whether and when guidelines like these should become requirements. For now, we will consider the scientific reporting of sex- and gender-related findings an important element of the papers we consider for publication. Our research needs to reflect that we treat both men and women, and that both are equally entitled to the benefits of care based on good, applicable evidence.

Regulation of mesenchymal stem cell chondrogenesis by glucose through protein kinase C/transforming growth factor signaling

OBJECTIVE Effective induction of human mesenchymal stem cell (hMSC) differentiation for regenerative medicine applications remains a great challenge. While much research has studied hMSC activity during differentiation, it is unclear whether pre-differentiation culture can modulate differentiation capacity. We investigate the effect of glucose concentration in pre-differentiation/expansion culture on modulating chondrogenic capacity of hMSCs, and explore the underlying molecular mechanism. DESIGN The extent of chondrogenesis of hMSCs previously cultured with different concentrations of glucose was evaluated. Transforming growth factor-beta (TGF-β) signaling molecules and protein kinase C (PKC) were analyzed to identify the role of these molecules in the regulation of glucose on chondrogenesis. In addition, hMSCs in high-glucose expansion culture were treated with the PKC inhibitor to modulate the activity of PKC and TGF-β signaling molecules. RESULTS High-glucose maintained hMSCs were less chondrogenic than low-glucose maintained cells upon receiving differentiation signals. Interestingly, we found that high-glucose culture increased the phosphorylation of PKC and expression of type II TGF-β receptor (TGFβRII) in pre-differentiation hMSCs. However, low-glucose maintained hMSCs became more responsive to chondrogenic induction with increased PKC activation and TGFβRII expression than high-glucose maintained hMSCs during differentiation. Inhibiting the PKC activity of high-glucose maintained hMSCs during expansion culture upregulated the TGFβRII expression of chondrogenic cell pellets, and enhanced chondrogenesis. CONCLUSION Our findings demonstrate the effect of glucose concentration on regulating the chondrogenic capability of pre-differentiation hMSCs, and provide insight into the mechanism of how glucose concentration regulates PKC and TGF-β signaling molecules to prime pre-differentiation hMSCs for subsequent chondrogenesis.