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Introduction The COVID-19 pandemic has affected and is still deeply affecting all aspects of public life. World governments have been forced to enact restrictive measures to stem the contagion which have led to a decrease in the movement of people within national territory and to a redirection of health care resources with a suspension of non-urgent procedures. In Italy, a lockdown was imposed from March 9 th to May 3 rd , 2020. As a result, a significant reduction in the overall operative volume of orthopedic trauma was expected, but it was not possible to predict a similar trend regarding fragility fractures of the proximal femur in the elderly. Methods The aim of this paper was to examine the impact of COVID-19 on the operating volume for trauma surgeries and to determine how the pandemic affected the management of fragility hip fractures (FHFs) in non-COVID patients at a single Institution. Results The first result was a statistically significant reduction in the overall operative volume of orthopedic trauma during the period of the first lockdown and an increase in the mean age of patients undergoing surgery, as expected. As regard to the second aim, the incidence of FHFs remained almost unchanged during the periods analysed. The population examined were superimposable in terms of demographics, comorbidities, type of fracture, peri-operative complications, percentage of operations performed within 48 hours from hospitalization and 1-year outcome. Discussion Our results are in line with those already present in the Literature. Conclusions Our study revealed a significant impact of the restrictive anti-contagion measures on the overall orthopedic surgical volume, but, at the same time, we could affirm that the pandemic did not affect the management of FHFs in non-COVID patients, and their results.

Introduction. The ever-expanding indications for total hip arthroplasty are leading to more implants being placed in younger as well as in older patients with high functional demand. Also, prolonged life expectancy is contributing to an overall increment of periprosthetic femoral fractures. The Vancouver classification has been the most used for guiding the surgeon choice since its proposal in 1995. Fractures occurring over a hip femoral implant can be divided into intra-operative and post-operative PFFs, and their treatment depends on factors that may severely affect the outcome: level of fracture, implant stability, quality of bone stock, patients’ functional demand, age and comorbidities, and surgeon expertise. There are many different treatment techniques available which include osteosynthesis and revision surgery or a combination of both. The goals of surgical treatment are patients’ early mobilization, restoration of anatomical alignment and length with a stable prosthesis and maintenance of bone stock. Significance. The aim of this review is to describe the state-of-the-art treatment and outcomes in the management of PFFs. We performed a systematic literature review of studies reporting on the management of PFFs around hip stems and inter-prosthetic fractures identifying 45 manuscripts eligible for the analysis. Conclusions. PFFs present peculiar characteristic that must be considered and special features that must be addressed. Their management is complex due to the extreme variability of stem designs, the possibility of having cemented or uncemented stems, the difficulty in identifying the “real” level of the fracture and the actual stability of the stem. As a result, the definition of a standardized treatment is unlikely, thereby high expertise is fundamental for the surgical management of PPFs, so this kind of fractures should be treated only in specialized centres with both high volume of revision joint arthroplasty and trauma surgery.

Introduction An increasing number of patients is annually undergoing total hip arthroplasty (THA), and a significant proportion of these patients are elderly and consequently at a higher risk of complications because of age, osteoporosis, and medical comorbidities. Periprosthetic femoral fractures (PFFs) are one of the worst complications of THA associated with high rates of unfavorable prognosis. Besides, in the last decade, a new independent disease entity called “atypical femoral fracture” (AFF) has been identified and defined by the American Society for Bone and Mineral Research (ASBMR) task force. Some PFFs present clinical history and radiographic aspect consistent with an AFF, meeting the ASBMR criteria for the diagnosis of AFF except that PFFs by themselves are an exclusion criterion for AFF. However, there is an increasing number of published studies suggesting that periprosthetic atypical femoral fractures (PAFFs) exist and should not be excluded by definition. Significance Nowadays, although there is an increasing interest in PAFFs, there are still very few studies published on the topic and a lack of consensus regarding their treatment. This narrative literature review aims to introduce this new emerging topic to a wider readership describing the characteristics of PAFFs and the state-of-the-art in their management. Conclusions Many authors agree that PAFFs should be considered as a subgroup of PFFs that have atypical characteristics; they also show a significant correlation with prolonged bisphosphonate use. A correct diagnosis is paramount for proper treatment of the disease that requires both surgical and medical actions to be taken.

Introduction There is lack of consensus regarding best operative fixation strategy for periprosthetic femoral fractures (PFFs) around a stable stem. Evidence exists that some patterns of fracture around a stable stem are better treated with revision surgery than with standard fixation. Anyway, a more aggressive surgical procedure together with medical treatment could allow for stem retention, and reduced risk of nonunion/hardware failure, even in these cases. Significance This paper is placed in a broader context of lack of studies on the matter, and its aim is to shed some light on the management of PFFs around a stable stem, when peculiar mechanical and biological aspects are present. Results Based on our casuistry in the treatment of nonunions after PFF successfully treated with original stem retention, and on review of Literature about risk factors for fixation failure, an algorithm is proposed that can guide in choosing the ideal surgical technique even for first-time PFFs with a stable stem, without resorting to revision. Mechanical (major and minor) and biological (local and systemic) factors that may influence fracture healing, leading to nonunion and hardware failure, and subsequent need for re-operation, are considered. The proposed surgical technique consists of rigid fixation with absolute stability (using a plate and structural allograft) plus local biological support (structural allograft and autologous bone marrow concentrate over a platelet-rich plasma-based scaffold) at fracture site. Systemic anabolic treatment (Teriparatide) is also administered in the post-operative period. Conclusion Mechanical factors are not the only issues to be considered when choosing the surgical approach to PFFs over a stable stem. Systemic and local biological conditions should be taken into account, as well. A therapeutic algorithm is proposed, given the prosthetic stem to be stable, considering mechanical and biological criteria.

In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair.

Hydroxytyrosol (HT), a phenolic compound mainly derived from olives, has been proposed as a nutraceutical useful in prevention or treatment of degenerative diseases. In the present study we have evaluated the ability of HT to counteract the appearance of osteoarthritis (OA) features in human chondrocytes. Pre-treatment of monolayer cultures of chondrocytes with HT was effective in preventing accumulation of reactive oxidant species (ROS), DNA damage and cell death induced by H2O2 exposure, as well as the increase in the mRNA level of pro-inflammatory, matrix-degrading and hypertrophy marker genes, such as iNOS, COX-2, MMP-13, RUNX-2 and VEGF. HT alone slightly enhanced ROS production, but did not enhance cell damage and death or the expression of OA-related genes. Moreover HT was tested in an in vitro model of OA, i.e. three-dimensional micromass cultures of chondrocytes stimulated with growth-related oncogene α (GROα), a chemokine involved in OA pathogenesis and known to promote hypertrophy and terminal differentiation of chondrocytes. In micromass constructs, HT pre-treatment inhibited the increases in caspase activity and the level of the messengers for iNOS, COX-2, MMP-13, RUNX-2 and VEGF elicited by GROα. In addition, HT significantly increased the level of SIRT-1 mRNA in the presence of GROα. In conclusion, the present study shows that HT reduces oxidative stress and damage, exerts pro-survival and anti-apoptotic actions and favourably influences the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features.

BACKGROUND: In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β. METHODOLOGY/PRINCIPAL FINDINGS: The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13. CONCLUSIONS/SIGNIFICANCE: Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.

Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage.

Mesenchymal stromal cells are emerging as an extremely promising therapeutic agent for tissue regeneration due to their multi-potency, immune-modulation and secretome activities, but safety remains one of the main concerns, particularly when in vitro manipulation, such as cell expansion, is performed before clinical application. Indeed, it is well documented that in vitro expansion reduces replicative potential and some multi-potency and promotes cell senescence. Furthermore, during in vitro aging there is a decrease in DNA synthesis and repair efficiency thus leading to DNA damage accumulation and possibly inducing genomic instability. The European Research Project ADIPOA aims at validating an innovative cell-based therapy where autologous adipose stromal cells (ASCs) are injected in the diseased articulation to activate regeneration of the cartilage. The primary objective of this paper was to assess the safety of cultured ASCs. The maintenance of genetic integrity was evaluated during in vitro culture by karyotype and microsatellite instability analysis. In addition, RT-PCR array-based evaluation of the expression of genes related to DNA damage signaling pathways was performed. Finally, the senescence and replicative potential of cultured cells was evaluated by telomere length and telomerase activity assessment, whereas anchorage-independent clone development was tested in vitro by soft agar growth. We found that cultured ASCs do not show genetic alterations and replicative senescence during the period of observation, nor anchorage-independent growth, supporting an argument for the safety of ASCs for clinical use.

Human mesenchymal stem cells (hMSCs) are pluripotent adult stem cells capable of being differentiated into osteoblasts, adipocytes, and chondrocytes. The osteogenic differentiation of hMSCs is regulated either by systemic hormones or by local growth factors able to induce specific intracellular signal pathways that modify the expression and activity of several transcription factors. Runt-related transcription factor 2 (Runx2) and Wnt signaling-related molecules are the major factors critically involved in the osteogenic differentiation process by hMSCs, and SRY-related high-mobility-group (HMG) box transcription factor 9 (SOX9) is involved in the chondrogenic one. hMSCs have generated a great interest in the field of regenerative medicine, particularly in bone regeneration. In this paper, we focused our attention on the molecular mechanisms involved in osteogenic and chondrogenic differentiation of hMSC, and the potential clinical use of hMSCs in osteoarticular pediatric disease characterized by fracture nonunion and pseudarthrosis.

The non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy.IKKα expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKα(f/f):CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant.IKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. Τηυσ, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

The activation of the NF-?B pathway by pro-inflammatory cytokines, such as tumor necrosis factor-a (TNFa), can be an important contributor for the re-programming of chondrocyte gene expression, thereby making it a therapeutic target in articular diseases. To search for new approaches to limit cartilage damage, we investigated the requirement of polyamines for NF-?B activation by TNFa in human C-28/I2 chondrocytes, using a-difluoromethylornithine (DFMO), a specific polyamine biosynthesis inhibitor. The NF-?B pathway was dissected by using pharmacological inhibitors or by expressing a transdominant I?Ba super repressor. Treatment of C-28/I2 chondrocytes with TNFa resulted in a rapid enhancement of nuclear localization and DNA binding activity of the p65 NF-?B subunit. TNFa also increased the level and extracellular release of interleukin-8 (IL-8), a CXC chemokine that can have a role in arthritis, in an NF-?B-dependent manner. Pre-treatment of chondrocytes with DFMO, while causing polyamine depletion, significantly reduced NF-?B DNA binding activity. Moreover, DFMO also decreased IL-8 production without affecting cellular viability. Restoration of polyamine levels by the co-addition of putrescine circumvented the inhibitory effects of DFMO. Our results show that the intracellular depletion of polyamines inhibits the response of chondrocytes to TNFa by interfering with the DNA binding activity of NF-?B. This suggests that a pharmacological and/or genetic approach to deplete the polyamine pool in chondrocytes may represent a useful way to reduce NF-?B activation by inflammatory cytokines in arthritis without provoking chondrocyte apoptosis. 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Chemokines are involved in a number of inflammatory pathologies and some of them show a pivotal role in the modulation of osteoclast development. Therefore, we evaluated the role of CXCL12 chemokine on osteoclast differentiation and function and we analyzed its expression on synovial and bone tissue biopsies from rheumatoid arthritis (RA) patients. Osteoclasts were obtained by 7 days in vitro differentiation with RANKL and M-CSF of CD11b positive cells in the presence or absence of CXCL12. The total number of osteoclast was analyzed by Tartrate-resistant acid phosphatase (TRAP)-staining and bone-resorbing activity was assessed by pit assay. MMP-9 and TIMP-1 release was evaluated by ELISA assay. CXCL12 expression on biopsies from RA patients was analyzed by immunohistochemistry. Osteoclasts obtained in the presence of CXCL12 at 10 nM concentration displayed a highly significant increase in bone-resorbing activity as measured by pit resorption assay, while the total number of mature osteoclasts was not affected. The increased resorption is associated with overexpression of MMP-9. Immunostaining for CXCL12 on synovial and bone tissue biopsies from both rheumatoid arthritis (RA) and osteoarthritis (OA) samples revealed a strong increase in the expression levels under inflammatory conditions. CXCL12 chemokine showed a clear activating role on mature osteoclast by inducing bone-resorbing activity and specific MMP-9 enzymatic release. Moreover, since bone and synovial biopsies from RA patients showed an elevated CXCL12 expression, these findings may provide useful tools for achieving a full elucidation of the complex network that regulates osteoclast function in course of inflammatory diseases. J. Cell. Physiol. 199: 244–251, 2004© 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Cartilage homeostasis dysregulation during osteoarthritis (OA) has been linked to an increased rate of apoptosis of chondrocytes, the only cell type resident in the cartilage. In addition, the CD95–CD95 ligand (the Fas system) has emerged as one of the major pathways of cell death in the cartilage. We undertook the present study to investigate the role of interferon-γ (IFNγ) in the regulation of the Fas system by analyzing the modulation of intracellular signaling molecules (FLICE inhibitory protein [FLIP] and caspases 3 and 8) in primary cultures of human OA chondrocytes. CD95-induced apoptotic death of human OA chondrocytes was analyzed in the presence or absence of IFNγ using cell death immunoassay for apoptosis, real-time polymerase chain reaction for FLIP and caspase 8 expression, Western blotting for FLIP, and proteolytic activity for caspases 3 and 8. CD95-induced apoptotic death of human OA chondrocytes was strongly counteracted by IFNγ treatment, although the surface expression of CD95 was slightly up-regulated by this cytokine. The messenger RNA (mRNA) expression of FLIP and caspase 8, mediators involved in CD95 signaling, revealed that FLIP expression in human OA chondrocytes was significantly up-regulated (2-fold increase) by IFNγ treatment. Moreover, the FLIP:caspase 8 mRNA ratio increased significantly. FLIP up-regulation by IFNγ was confirmed at the protein level. Caspase 8 and caspase 3 proteolytic activities, both induced in these cells by stimulation with anti-CD95, were also significantly down-modulated by IFNγ. These findings suggest that IFNγ impairs CD95-mediated signaling and apoptotic death in human chondrocytes. Its mechanism of action involves down-regulation of caspase 8 and caspase 3 activities and increased expression of the antiapoptotic protein FLIP, suggesting an interesting mechanism for the inhibition of chondrocyte apoptosis. [ABSTRACT FROM AUTHOR]

To extend the study of the chemokine receptor repertoire on human chondrocytes to receptors with reported housekeeping functions (CXCR3, CXCR4, CXCR5, and CCR6) and to evaluate whether ligands of these receptors play a role in chondrocyte phenotype modulation and proliferation. Chemokine receptor expression was determined by flow cytometry. Subcultures of chondrocytes were collected and fixed at confluence or during the exponential phase of growth and analyzed for chemokine receptor modulation. The effects of chemokines on isolated cells as well as chondrocytes cultured within an intact extracellular matrix were investigated. Isolated human chondrocytes were stimulated with 100 nM chemokines (monokine induced by interferon-γ, stromal cell–derived factor 1α [SDF-1α], B cell–attracting chemokine 1 [BCA-1], or macrophage inflammatory protein 3α), and conditioned media were assessed for matrix-degrading enzyme contents (matrix metalloproteinases [MMPs] 1, 3, and 13, and N-acetyl-β-D-glucosaminidase [NAG]). Cell proliferation and phenotype modulation were evaluated by bromodeoxyuridine incorporation and cathepsin B production. Induction of cell proliferation was assessed in cartilage explants by immunodetection of the proliferation-associated antigen S100A4. CXCR3, CXCR4, CXCR5, and CCR6 were detected on human chondrocytes. CXCR3 and CXCR4 expression was increased in exponentially growing chondrocyte subcultures. Ligands of all receptors enhanced the release of MMPs 1, 3, and 13. Release of NAG and cathepsin B was significantly higher in chemokine-stimulated cultures than in unstimulated cultures. SDF-1α and BCA-1 also induced DNA synthesis and chondrocyte proliferation, as was shown by the up-regulation of S100A4 in cartilage explants as well. Our findings extend the repertoire of functional responses elicited by the activity of chemokines on chondrocytes and open new avenues in our understanding of the control of chondrocyte differentiation status by chemokines and their receptors. [ABSTRACT FROM AUTHOR]

O trevo vermelho é uma leguminosa forrageira de clima temperado que apresenta bom potencial de produção de matéria seca e qualidade de forragem, sendo indicado para complementar a dieta dos animais durante a estação fria do ano no sul do Brasil. O objetivo do trabalho foi, por meio da realização de uma análise de trilha para rendimento de sementes em trevo vermelho, obter informações básicas para utilização em programas de melhoramento e/ou estratégias de manejo que levem à maior produção de sementes e, conseqüentemente, maior ressemeadura natural, proporcionando condições para maior persistência da espécie na pastagem. Foram analisadas as seguintes características: rendimento de sementes, número de hastes por planta, número de inflorescências por planta, número de inflorescências por haste e peso de mil sementes. A variável que mais se correlacionou com o rendimento de sementes foi o número de inflorescências por planta (r = 0,710), sendo que essa correlação se deu praticamente apenas pelo efeito direto da mesma. As demais variáveis apresentaram menores valores de correlação com o rendimento de sementes (r entre 0,175 e 0,578), com efeitos diretos reduzidos ou negativos sobre essa última característica. Portanto, o número de inflorescências por planta mostrou-se como a principal característica a ser considerada na busca de maior produção de sementes em trevo vermelho.

O objetivo deste trabalho foi avaliar a variabilidade de características agronômicas de uma população de trevo vermelho (Trifolium pratense L.) tais como: taxa de crescimento em altura nos períodos vegetativo e reprodutivo, taxa de crescimento em diâmetro no períodos vegetativo e reprodutivo, rendimento de matéria seca por planta, produção de sementes e seus componentes (hastes/planta, inflorescências/plant