Engineered Cartilage Maturation Regulates Cytokine Production and Interleukin-1β Response

Background: Because the injured joint has an actively inflammatory environment, the survival and repair potential of cartilage grafts may be influenced by inflammatory processes. Understanding the interactions of those processes with the graft may lead to concepts for pharmacologic or surgical solutions allowing improved cartilage repair. Questions/purposes: We asked whether the maturation level of cartilaginous tissues generated in vitro by expanded human articular chondrocytes (HACs) modulate (1) the spontaneous production of cytokines and (2) the response to interleukin (IL)-1β. Methods: Twelve pellets/donor prepared with monolayer-expanded HACs (n=6 donors) were evaluated at six different culture times for mRNA expression (n=72) and spontaneous baseline release of monocyte chemoattractant protein (MCP)-1, IL-8, and transforming growth factor (TGF)-β1 (n=72). We cultured 24 pellets/donor from each of four donors for 1 or 14days (defined as immature and mature, respectively) and exposed the pellets to IL-1β for 3days. MCP-1, IL-8, TGF-β1, and metalloprotease (MMP)-1 and MMP-13 were quantified in pellets and culture supernatants. Results: By increasing culture time, the spontaneous release of IL-8 and MCP-1 decreased (12.0- and 5.5-fold, respectively), whereas that of TGF-β1 increased (5.4-fold). As compared with immature pellets, mature pellets responded to IL-1β by releasing lower amounts of MMP-1 (2.9-fold) and MMP-13 (1.7-fold) and increased levels of IL-8, MCP-1, and TGF-β1 (1.5-, 5.0-, and 7.5-fold, respectively). IL-8 and MCP-1 promptly returned to baseline on withdrawal of IL-1β. Conclusions: Our observations suggest more mature cartilaginous tissues are more resistant to IL-1β exposure and can activate chemokines required to initiate tissue repair processes. Clinical Relevance: The implantation of more mature cartilaginous tissues might provide superior graft survival and improve/accelerate cartilage repai

Growth factors for clinical-scale expansion of human articular chondrocytes : Relevance for automated bioreactor systems

The expansion of chondrocytes in automated bioreactors for clinical use requires that a relevant number of cells be generated, starting from variable initial seeding densities in one passage and using autologous serum. We investigated whether the growth factor combination transforming growth factor beta 1/fibroblast growth factor 2/platelet-derived growth factor BB (TFP), recently shown to enhance the proliferation capacity of human articular chondrocytes (HACs), allows the efficiency of chondrocyte use to be increased at different seeding densities and percentages of human serum (HS). HACs were seeded at 1,000, 5,000, and 10,000 celIS/cm(2) in medium containing 10 bovine serum or 10,000 cells/cm(2) with 1 chondrogenic capacity of post-expanded HACs was then assessed in pellet cultures. Expansion with TFP allowed a sufficient number of HACs to be obtained in one passage even at the lowest seeding density and HS percentage and variability in cartilage-forming capacity of HACs expanded under the different conditions to be reduced. Instead, larger variations and insufficient yields were found in the absence of TFP. By allowing large numbers of cells to be obtained, starting from a wide range of initial seeding densities and HS percentages, the use of TFP may represent a viable solution for the efficient expansion of HACs and addresses constraints of automated clinical bioreactor systems

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Influence of chondrocytes differentiation stage on the capacity to generate cartilaginous tissue in vitro

Cartilage lesions resulting from trauma or degenerative diseases are one of the major factors leading to joint disease and disability. Articular cartilage being an avascular tissue has limited capacity for self repair. To overcome these limitations, tissue engineering techniques have emerged as an innovative field of research with the potential to recreate three dimensional cartilaginous tissues. Such approach generally rely on the expansion of a limited population of chondrocytes derived from a small cartilage biopsy, intrinsically associated with cellular de-differentiation (108), and on the ability of the expanded cells to efficiently and reproducibly re-differentiate and generate cartilaginous tissue. The objective of my research was to study different clinically relevant aspects of the biology of human articular chondrocytes, related to different phases in the process of engineering of a functional cartilage tissue (Figure 4). In particular, my thesis was aimed at determining: - whether the supplementation of growth factors enhancing chondrocyte proliferation and re-differentiation capacity allows a reproducible and efficient clinical-scale expansion of human articular chondrocytes (HAC) starting from variable initial seeding densities and using low percentages of human serum (Study 1; Francioli et al., Tissue Eng (2007)); - whether the quality of cartilaginous tissues generated by HAC on three-dimensional biomimetic scaffolds can be enhanced by direct expansion on the scaffold, as compared to standard growth on plastic, or by increasing cell seeding density (Study 2; Francioli et al., Tissue Eng (submitted)); - how the extent of maturation of HAC-based cartilaginous tissues modulates the profile of chemokine production and the inflammatory/catabolic response to IL-1β (Study 3; Francioli et al., Clin Orthop Relat Res (in preparation for submission))

L'Uso di Culture Cellulari nei Test di Tossicità Richiesti per Legge: Anadri Tossicologici Relativi a Metalli in Tracce (Tesi presentata ...)

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra

Engineered cartilage maturation regulates cytokine production and interleukin-1beta response

BACKGROUND: Because the injured joint has an actively inflammatory environment, the survival and repair potential of cartilage grafts may be influenced by inflammatory processes. Understanding the interactions of those processes with the graft may lead to concepts for pharmacologic or surgical solutions allowing improved cartilage repair. QUESTIONS/PURPOSES: We asked whether the maturation level of cartilaginous tissues generated in vitro by expanded human articular chondrocytes (HACs) modulate (1) the spontaneous production of cytokines and (2) the response to interleukin (IL)-1beta. METHODS: Twelve pellets/donor prepared with monolayer-expanded HACs (n = 6 donors) were evaluated at six different culture times for mRNA expression (n = 72) and spontaneous baseline release of monocyte chemoattractant protein (MCP)-1, IL-8, and transforming growth factor (TGF)-beta1 (n = 72). We cultured 24 pellets/donor from each of four donors for 1 or 14 days (defined as immature and mature, respectively) and exposed the pellets to IL-1beta for 3 days. MCP-1, IL-8, TGF-beta1, and metalloprotease (MMP)-1 and MMP-13 were quantified in pellets and culture supernatants. RESULTS: By increasing culture time, the spontaneous release of IL-8 and MCP-1 decreased (12.0- and 5.5-fold, respectively), whereas that of TGF-beta1 increased (5.4-fold). As compared with immature pellets, mature pellets responded to IL-1beta by releasing lower amounts of MMP-1 (2.9-fold) and MMP-13 (1.7-fold) and increased levels of IL-8, MCP-1, and TGF-beta1 (1.5-, 5.0-, and 7.5-fold, respectively). IL-8 and MCP-1 promptly returned to baseline on withdrawal of IL-1beta. CONCLUSIONS: Our observations suggest more mature cartilaginous tissues are more resistant to IL-1beta exposure and can activate chemokines required to initiate tissue repair processes. CLINICAL RELEVANCE: The implantation of more mature cartilaginous tissues might provide superior graft survival and improve/accelerate cartilage repair

Effect of three-dimensional expansion and cell seeding density on the cartilage-forming capacity of human articular chondrocytes in type II collagen sponges

Chondrocytes for tissue engineering strategies are typically expanded in monolayer (2D), leading to cell dedifferentiation but allowing to generate large cell numbers for seeding into scaffolds. Direct chondrocyte culture in scaffolds, instead, may support better maintenance of the differentiated phenotype but reduce the extent of proliferation and thus the resulting cell density. This study investigates whether the quality of cartilaginous tissues generated in vitro by human articular chondrocytes (HAC) on type II collagen sponges is enhanced (1) by direct expansion on the scaffolds (3D), as compared with standard 2D, or (2) by increasing cell seeding density, which in turn requires extensive 2D expansion. Three-dimensional expansion of HAC on the scaffolds, as compared with 2D expansion for the same number of doublings, better maintained the chondrocytic phenotype of the expanded cells (13.7-fold higher levels of type II collagen mRNA) but did not enhance their accumulation of glycosaminoglycan (GAG) following chondrogenic culture. Instead, increasing the HAC seeding density in the scaffolds (from 25 × 10(3) to 66 × 10(3) cells/mm(3)) significantly improved chondrogenesis (up to 3.3-fold higher GAG accumulation and up to 9.3-fold higher type II collagen mRNA), even if seeded cells had to be expanded and dedifferentiated more extensively in 2D to reach the required cell numbers. This study indicates that, under the specific conditions tested, a high-seeding density of HAC in 3D scaffolds is more critical for the generation of cartilaginous constructs than the stage of cell differentiation reached following expansion

Design of graded biomimetic osteochondral composite scaffolds

With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues. A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their self-assembling. The resulting mineral phase was non-stoichiometric and amorphous, resembling chemico-physical features of newly deposited, natural bone matrix. A graded material was then generated, consisting of (i) a lower layer of the developed biomineralized collagen, corresponding to the subchondral bone, (ii) an upper layer of hyaluronic acid-charged collagen, mimicking the cartilaginous region, and (iii) an intermediate layer of the same nature as the biomineralized collagen, but with a lower extent of mineral, resembling the tidemark. The layers were stacked and freeze-dried to obtain an integrated monolithic composite. Culture of the material for 2 weeks after loading with articular chondrocytes yielded cartilaginous tissue formation selectively in the upper layer. Conversely, ectopic implantation in nude mice of the material after loading with bone marrow stromal cells resulted in bone formation which remained confined within the lower layer. in conclusion, we developed a composite material with cues which are biomimetic of an osteochondral tissue and with the capacity to differentially support cartilage and bone tissue generation. The results warrant testing of the material as a substitute for the repair of osteochondral lesions in orthotopic animal models

Importance of Baseline Prognostic Factors With Increasing Time Since Initiation of Highly Active Antiretroviral Therapy: Collaborative Analysis of Cohorts of HIV-1-Infected Patients

Background: The extent to which the prognosis for AIDS and death of patients initiating highly active antiretroviral therapy (HAART) continues to be affected by their characteristics at the time of initiation (baseline) is unclear. Methods: We analyzed data on 20,379 treatment-naive HIV-1- infected adults who started HAART in 1 of 12 cohort studies in Europe and North America (61,798 person-years of follow-up, 1844 AIDS events, and 1005 deaths). Results: Although baseline CD4 cell count became less prognostic with time, individuals with a baseline CD4 count 350 cells/μL (hazard ratio for AIDS = 2.3, 95% confidence interval [CI]: 1.0 to 2.3; mortality hazard ratio = 2.5, 95% CI: 1.2 to 5.5, 4 to 6 years after starting HAART). Rates of AIDS were persistently higher in individuals who had experienced an AIDS event before starting HAART. Individuals with presumed transmission by means of injection drug use experienced substantially higher rates of AIDS and death than other individuals throughout follow-up (AIDS hazard ratio = 1.6, 95% CI: 0.8 to 3.0; mortality hazard ratio = 3.5, 95% CI: 2.2 to 5.5, 4 to 6 years after starting HAART). Conclusions: Compared with other patient groups, injection drug users and patients with advanced immunodeficiency at baseline experience substantially increased rates of AIDS and death up to 6 years after starting HAART