PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation.

VEGF induces normal or aberrant angiogenesis depending on its dose in the microenvironment around each producing cell in vivo. This transition depends on the balance between VEGF-induced endothelial stimulation and PDGF-BB-mediated pericyte recruitment, and co-expression of PDGF-BB normalizes aberrant angiogenesis despite high VEGF doses. We recently found that VEGF over-expression induces angiogenesis in skeletal muscle through an initial circumferential vascular enlargement followed by longitudinal splitting, rather than sprouting. Here we investigated the cellular mechanism by which PDGF-BB co-expression normalizes VEGF-induced aberrant angiogenesis. Monoclonal populations of transduced myoblasts, expressing similarly high levels of VEGF alone or with PDGF-BB, were implanted in mouse skeletal muscles. PDGF-BB co-expression did not promote sprouting and angiogenesis that occurred through vascular enlargement and splitting. However, enlargements were significantly smaller in diameter, due to a significant reduction in endothelial proliferation, and retained pericytes, which were otherwise lost with high VEGF alone. A time-course of histological analyses and repetitive intravital imaging showed that PDGF-BB co-expression anticipated the initiation of vascular enlargement and markedly accelerated the splitting process. Interestingly, quantification during in vivo imaging suggested that a global reduction in shear stress favored the initiation of transluminal pillar formation during VEGF-induced splitting angiogenesis. Quantification of target gene expression showed that VEGF-R2 signaling output was significantly reduced by PDGF-BB co-expression compared to VEGF alone. In conclusion, PDGF-BB co-expression prevents VEGF-induced aberrant angiogenesis by modulating VEGF-R2 signaling and endothelial proliferation, thereby limiting the degree of circumferential enlargement and enabling efficient completion of vascular splitting into normal capillary networks despite high VEGF doses.

Governance of hospital alliances: lessons learnt from 6 hospital and non-hospital cases.

Hospital alliances can be a solution for the coordination that is required between providers as a result of the increasing fragmentation in health-care services. 3 hospital and 3 non-hospital alliances were studied with a qualitative case study methodology, to find common, or specific, practices with regards to the management of alliances. Striking similarities prevailed, hospital alliances are not unique and common principles of coordination were identified that appear pivotal for successful alliance management.

Perspective on the evolution of cell-based bone tissue engineering strategies.

Despite the compelling clinical needs in enhancing bone regeneration and the potential offered by the field of tissue engineering, the adoption of cell-based bone graft substitutes in clinical practice is limited to date. In fact, no study has yet convincingly demonstrated reproducible clinical performance of tissue-engineered implants and at least equivalent cost-effectiveness compared to the current treatment standards. Here, we propose and discuss how tissue engineering strategies could be evolved towards more efficient solutions, depicting three different experimental paradigms: (i) bioreactor-based production; (ii) intraoperative manufacturing, and (iii) developmental engineering. The described approaches reflect the need to streamline graft manufacturing processes while maintaining the potency of osteoprogenitors and recapitulating the sequence of biological steps occurring during bone development, including vascularization. The need to combine the assessment of efficacy of the different strategies with the understanding of their mechanisms of action in the target regenerative processes is highlighted. This will be crucial to identify the necessary and sufficient set of signals that need to be delivered at the injury or defect site and should thus form the basis to define release criteria for reproducibly effective engineered bone graft substitutes.

[From pay-for-effort to pay-for-performance--an analysis of the Swiss health care system with focus on the inpatient sector]. / Von der Kostenerstattung zur leistungs- und ergebnisorientierten Vergütung--Eine Analyse des schweizerischen Gesundheitswesens mit Fokus auf dem stationären Bereich.

INTRODUCTION: The Swiss health care system is facing the implementation of lump compensation in the form of diagnosis related groups from 2010 on. In addition there is an increasing discussion about the quality of health care in the media. We have analyzed current remuneration in Swiss health care and their steering effects on providers in order to deduct future developments in Swiss health care remuneration. METHODS: Based on the remuneration contracts and tariff regularities at the Basel University Hospital we conducted an internet and literature search. The identified Swiss remuneration systems were classified after remuneration scales and remuneration item using a typology of performance-related remuneration systems. The steering effects of the remuneration systems on the providers were deducted. RESULTS: Remuneration scales can be classified in <>, <> or <>. Remuneration items can be classified in <>, <> or <>. Remuneration systems can lead to increased or decreased services or to patient selection. In the Swiss health care system we find a trend away from traditional <> to <> remuneration systems. In that context diagnosis related groups are identified as an intermediate step in the development of remuneration systems. CONCLUSIONS: Future developments of medical remuneration in terms of a consideration of quality of medical performance and negotiated costs seem likely in Switzerland in the long term. Both, economics and quality should be considered adequately in a health care remuneration system.

A single exposure to social isolation in domestic piglets activates behavioural arousal, neuroendocrine stress hormones, and stress-related gene expression in the brain.

Stressful early life events can have short- and long-term effects on neuroendocrine and behavioural mechanisms of adaptation. Here, we investigated the effects of a single social isolation (4 h) of domestic piglets on both behavioural alterations in open-field tests and modifications in the expression of genes regulating glucocorticoid response in stress-related brain regions at 7, 21 or 35 days of age. The mRNAs of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ss-hydroxysteroid dehydrogenase 1 and 2 (11ss-HSD1 and 11ss-HSD2) and c-fos were analysed by real-time RT-PCR in the hypothalamus, hippocampus and amygdala. The social isolation caused both elevated stress hormone concentrations (e.g. cortisol) and open-field reactivity (e.g. locomotion, vocalisation) compared to control piglets. The enhanced behavioural and neuroendocrine activity was associated with distinct changes in gene expression in the limbic system. The hypothalamic GR, MR and 11ss-HSD1 mRNA expressions and the hippocampal 11ss-HSD1 mRNA was significantly higher in isolated piglets, whereas in the amygdala social isolation caused a significant decrease in MR mRNA expression. Isolated piglets also displayed significantly higher c-fos mRNA expression, an estimate of neuronal activation, in hypothalamus and amygdala. The mRNA alterations as well as the behavioural and hormonal pattern show an effect of social isolation on days 7 and 21, but no effect on day 35. In conclusion, a single social isolation in piglets caused age-dependent neuroendocrine and behavioural changes that indicate increased arousal and experienced distress. The present results also suggest that psychosocial stress effects should be considered for the assessment of livestock handling practices with respect to health and welfare.

A novel implantation technique for engineered osteo-chondral grafts.

We present a novel method to support precise insertion of engineered osteochondral grafts by pulling from the bone layer, thereby minimizing iatrogenic damage associated with direct manipulation of the cartilage layer. Grafts were generated by culturing human expanded chondrocytes on Hyaff-11 meshes, sutured to Tutobone spongiosa cylinders. Through the bone layer, shaped to imitate the surface-contours of the talar dome, two sutures were applied: the first for anterograde implantation, to pull the graft into the defect, and the second for retrograde correction, in case of a too deep insertion. All grafts could be correctly positioned into osteochondral lesions created in cadaveric ankle joints with good fit to the surrounding cartilage. Implants withstood short-term dynamic stability tests applied to the ankle joint, without delamination or macroscopic damage. The developed technique, by allowing precise and stable positioning of osteochondral grafts without iatrogenic cartilage damage, is essential for the implantation of engineered tissues, where the cartilage layer is not fully mechanically developed, and could be considered also for conventional autologous osteochondral transplantation.

Intra-individual comparison of human ankle and knee chondrocytes in vitro: relevance for talar cartilage repair.

OBJECTIVE: As compared to knee chondrocytes (KC), talar chondrocytes (TC) have superior synthetic activity and increased resistance to catabolic stimuli. We investigated whether these properties are maintained after TC are isolated and expanded in vitro. METHODS: Human TC and KC from 10 cadavers were expanded in monolayer and then cultured in pellets for 3 and 14 days or in hyaluronan meshes (Hyaff-11) for 14 and 28 days. Resulting tissues were assessed biochemically, histologically, biomechanically and by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The proteoglycan and collagen synthesis rates in the pellets were also measured following exposure to Interleukin-1 beta (IL-1 beta). RESULTS: After 14 days of pellet culture, TC and KC expressed similar levels of type I collagen (CI) and type II collagen (CII) mRNA and the resulting tissues contained comparable amounts of glycosaminoglycans (GAG) and displayed similar staining intensities for CII. Also proteoglycan and collagen synthesis were similar in TC and KC pellets, and dropped to a comparable extent in response to IL-1 beta. Following 14 days of culture in Hyaff-11, TC and KC generated tissues with similar amounts of GAG and CI and CII. After 28 days, KC deposited significantly larger fractions of GAG and CII than TC, although the trend was not reflected in the measured biomechanical properties. CONCLUSION: After isolation from their original matrices and culture expansion, TC and KC displayed similar biosynthetic activities, even in the presence of catabolic stimuli. These in vitro data suggest a possible equivalence of TC and KC as autologous cell sources for the repair of talar cartilage lesions.

[Organization of clinical emergency units. Mission and environmental factors determine the organizational concept]. / Organisation der Notfallstation. Umfeld und Leistungsauftrag bestimmen das Organisationskonzept.

AIM: Mission and organization of emergency units were analysed to understand the underlying principles and concepts. METHODS: The recent literature (2000-2007) on organizational structures and functional concepts of clinical emergency units was reviewed. An organizational portfolio based on the criteria specialization (presence of medical specialists on the emergency unit) and integration (integration of the emergency unit into the hospital structure) was established. The resulting organizational archetypes were comparatively assessed based on established efficiency criteria (efficiency of resource utilization, process efficiency, market efficiency). RESULTS: Clinical emergency units differ with regard to autonomy (within the hospital structure), range of services and service depth (horizontal and vertical integration). The "specialization"-"integration"-portfolio enabled the definition of typical organizational patterns (so-called archetypes): profit centres primarily driven by economic objectives, service centres operating on the basis of agreements with the hospital board, functional clinical units integrated into medical specialty units (e.g., surgery, gynaecology) and modular organizations characterized by small emergency teams that would call specialists immediately after triage and initial diagnostic. CONCLUSIONS: There is no "one fits all" concept for the organization of clinical emergency units. Instead, a number of well characterized organizational concepts are available enabling a rational choice based on a hospital's mission and demand.

Cartilage tissue engineering using pre-aggregated human articular chondrocytes.

In this study, we first aimed at determining whether human articular chondrocytes (HAC) proliferate in aggregates in the presence of strong chondrocyte mitogens. We then investigated if the aggregated cells have an enhanced chondrogenic capacity as compared to cells cultured in monolayer. HAC from four donors were cultured in tissue culture dishes either untreated or coated with 1% agarose in the presence of TGFbeta-1, FGF-2 and PDGF-BB. Proliferation and stage of differentiation were assessed by measuring respectively DNA contents and type II collagen mRNA. Expanded cells were induced to differentiate in pellets or in Hyaff-11 meshes and the formed tissues were analysed biochemically for glycosaminoglycans (GAG) and DNA, and histologically by Safranin O staining. The amount of DNA in aggregate cultures increased significantly from day 2 to day 6 (by 3.2-fold), but did not further increase with additional culture time. Expression of type II collagen mRNA was about two orders of magnitude higher in aggregated HAC as compared to monolayer expanded cells. Pellets generated by aggregated HAC were generally more intensely stained for GAG than those generated by monolayer-expanded cells. Scaffolds seeded with aggregates accumulated more GAG (1.3-fold) than scaffolds seeded with monolayer expanded HAC. In conclusion, this study showed that HAC culture in aggregates does not support a relevant degree of expansion. However, aggregation of expanded HAC prior to loading into a porous scaffold enhances the quality of the resulting tissues and could thus be introduced as an intermediate culture phase in the manufacture of engineered cartilage grafts.

Engineered cartilage generated by nasal chondrocytes is responsive to physical forces resembling joint loading.

OBJECTIVE: To determine whether engineered cartilage generated by nasal chondrocytes (ECN) is responsive to different regimens of loading associated with joint kinematics and previously shown to be stimulatory of engineered cartilage generated by articular chondrocytes (ECA). METHODS: Human nasal and articular chondrocytes, harvested from 5 individuals, were expanded and cultured for 2 weeks into porous polymeric scaffolds. The resulting ECN and ECA were then maintained under static conditions or exposed to the following loading regimens: regimen 1, single application of cyclic deformation for 30 minutes; regimen 2, intermittent application of cyclic deformation for a total of 10 days, followed by static culture for 2 weeks; regimen 3, application of surface motion for a total of 10 days. RESULTS: Prior to loading, ECN constructs contained significantly higher amounts of glycosaminoglycan (GAG) and type II collagen compared with ECA constructs. ECN responded to regimen 1 by increasing collagen and proteoglycan synthesis, to regimen 2 by increasing the accumulation of GAG and type II collagen as well as the dynamic modulus, and to regimen 3 by increasing the expression of superficial zone protein, at the messenger RNA level and the protein level, as well as the release of hyaluronan. ECA constructs were overall less responsive to all loading regimens, likely due to the lower extracellular matrix content. CONCLUSION: Human ECN is responsive to physical forces resembling joint loading and can up-regulate molecules typically involved in joint lubrication. These findings should prompt future in vivo studies exploring the possibility of using nasal chondrocytes as a cell source for articular cartilage repair.

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes.

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

Bi-zonal cartilaginous tissues engineered in a rotary cell culture system.

In this study, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (HYAFF-11), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGFbeta3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution. In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.

Cartilage tissue engineering by expanded goat articular chondrocytes.

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.

Structural characterization and reliable biomechanical assessment of integrative cartilage repair.

Structural and functional characterization of integrative cartilage repair in controlled model systems can play a key role in the development of innovative strategies to improve the long-term outcome of many cartilage repair procedures. In this work, we first developed a method to reproducibly generate geometrically defined disk/ring cartilage composites and to remove outgrown fibrous layers which can encapsulate cartilaginous tissues during culture. We then used the model system to test the hypothesis that such fibrous layers lead to an overestimation of biomechanical parameters of integration at the disk/ring interface. Transmission electron microscopy images of the composites after 6 weeks of culture indicated that collagen fibrils in the fibrous tissue layer were well integrated into the collagen network of the cartilage disk and ring, whereas molecular bridging between opposing disk/ring cartilage surfaces was less pronounced and restricted to regions with narrow interfacial regions (< 2 microm). Stress-strain profiles generated from mechanical push-out tests for composites with the layers removed displayed a single and distinct peak, whereas profiles for composites with the layers left intact consisted of multiple superimposed peaks. As compared to composites with removed layers, composites with intact layers had significantly higher adhesive strengths (161+/-9 vs. 71+/-11 kPa) and adhesion energies (15.0+/-0.7 vs. 2.7+/-0.4 mJ/mm2). By combining structural and functional analyses, we demonstrated that the outgrowing tissue formed during in vitro culture of cartilaginous specimens should be eliminated in order to reliably quantify biomechanical parameters related to integrative cartilage repair.

Chondrogenesis of expanded adult human articular chondrocytes is enhanced by specific prostaglandins.

OBJECTIVE: To investigate the effects of the cyclooxygenase-2 (cox-2)-dependent prostaglandins D(2) (PGD(2)), E(2) (PGE(2)) and F(2)alpha (PGF(2)alpha) on the redifferentiation and cartilage matrix production of dedifferentiated articular chondrocytes. METHODS: Human articular chondrocytes from three adult donors were dedifferentiated by monolayer expansion and induced to redifferentiate by culture as 3D pellets in a defined serum-free medium containing TGF-beta(1) and dexamethasone, without or with further supplementation with PGD(2), PGE(2) or PGF(2)alpha. After 2 weeks, pellets were assessed histologically, immunohistochemically, biochemically and by real-time quantitative reverse transcriptase-polymerase chain reaction. RESULTS: All three PGs, but predominantly PGE(2), reduced the staining intensity of pellets for collagen type I, whereas PGD(2) and PGF(2)alpha increased the staining intensity of pellets for collagen type II and glycosaminoglycans (GAG). The GAG/DNA content of pellets was not affected by PGE(2) but was increased 1.5- and 2.1-fold by PGD(2) and PGF(2)alpha respectively. PGE(2) reduced the expression of collagen type I mRNA (9.0-fold), whereas PGD(2) and PGF(2)alpha increased the mRNA expression of collagen type II (6.2- and 4.1-fold respectively) and aggrecan (29.8- and 10.7-fold respectively). CONCLUSION: In contrast to PGE(2), PGD(2) and PGF(2)alpha enhanced chondrogenic differentiation and hyaline cartilage matrix deposition by expanded human articular chondrocytes, and could thus be used to improve in vitro or in vivo cartilage regeneration approaches based on these cells.

[Engineering and characterization of functional osteochondral replacement tissue]. / Engineering und Charakterisierung von funktionalem osteochondralem Ersatzgewebe.

Extensive osteochondral lesions require repair of the cartilage and underlying bone. We generated osteochondral repair tissue by tissue engineering. Standardized defects, 7 x 5 x 5 mm, were created in femoropatellar grooves of adult rabbits. Engineered cartilage, generated in vitro starting from chondrocytes and a biodegradable scaffold, was implanted using Collagraft as subchondral support. Cell-free implants, defects left empty, and unoperated knee joints served as controls. Explants were characterized morphologically and mechanically. Engineered cartilage implants were superior to cell-free implants and to natural healing of empty defects with respect to the histologic score and Young's modulus of the 6-month repair tissue. These data suggest that engineered cartilage can provide primary stability for the treatment of critical osteochondral defects.

Encapsulation into sterically stabilised liposomes enhances the immunogenicity of melanoma-associated Melan-A/MART-1 epitopes.

Tumour-associated antigens (TAA)-specific vaccination requires highly immunogenic reagents capable of inducing cytotoxic T cells (CTL). Soluble peptides are currently used in clinical applications despite an acknowledged poor immunogenicity. Encapsulation into liposomes has been suggested to improve the immunogenicity of discrete antigen formulations. We comparatively evaluated the capacity of HLA-A2.1 restricted Melan-A/MART-1 epitopes in soluble form (S) or following inclusion into sterically stabilised liposomes (SSL) to be recognised by specific CTL, to stimulate their proliferation and to induce them in healthy donors' peripheral blood mononuclear cells (PBMC), as well as in melanoma-derived tumour-infiltrating lymphocytes (TIL). HLA-A2.1(+), Melan-A/MART-1-NA-8 melanoma cells served as targets of specific CTL in 51Cr release assays upon pulsing by untreated or human plasma-treated soluble or SSL-encapsulated Melan-A/MART-1 27-35 (M27-35) or 26-35 (M26-35) epitopes. These reagents were also used to stimulate CTL proliferation, measured as 3H-thymidine incorporation, in the presence of immature dendritic cells (iDC), as antigen-presenting cells (APC). Induction of specific CTL upon stimulation with soluble or SSL-encapsulated peptides was attempted in healthy donors' PBMC or melanoma-derived TIL, and monitored by 51Cr release assays and tetramer staining. Na-8 cells pulsing with SSL M27-35 resulted in a five-fold more effective killing by specific CTL as compared with equal amounts of S M27-35. Encapsulation into SSL also provided a partial (50%) protection of M27-35 from plasma hydrolysis. No specific advantages regarding M26-35 were detectable in these assays. However, at low epitope concentrations (

Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes.

Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.