Identification of tissue-dependent proteins in knee OA synovial fluid.

OBJECTIVE: For many proteins from osteoarthritic synovial fluid, their intra-articular tissue of origin remains unknown. In this study we performed comparative proteomics to identify osteoarthritis-specific and joint tissue-dependent secreted proteins that may serve as candidates for osteoarthritis biomarker development on a tissue-specific basis. DESIGN: Protein secretomes of cartilage, synovium, Hoffa's fat pad and meniscus from knee osteoarthritis patients were determined using liquid chromatography tandem mass spectrometry, followed by label-free quantification. Validation of tissue-dependent protein species was conducted by ELISA on independent samples. Differential proteomes of osteoarthritic and non-osteoarthritic knee synovial fluids were obtained via similar proteomics approach, followed by ELISA validation. RESULTS: Proteomics revealed 64 proteins highly secreted from cartilage, 94 from synovium, 37 from Hoffa's fat pad and 21 from meniscus. Proteomic analyses of osteoarthritic vs non-osteoarthritic knee synovial fluid revealed 70 proteins with a relatively higher abundance and 264 proteins with a relatively lower abundance in osteoarthritic synovial fluid. Of the 70 higher abundance proteins, 23 were amongst the most highly expressed in the secretomes of a specific intra-articular tissue measured. Tissue-dependent release was validated for SLPI, C8, CLU, FN1, RARRES2, MATN3, MMP3 and TNC. Abundance in synovial fluid of tissue-dependent proteins was validated for IGF2, AHSG, FN1, CFB, KNG and C8. CONCLUSIONS: We identified proteins with a tissue-dependent release from intra-articular human knee OA tissues. A number of these proteins also had an osteoarthritis-specific abundance in knee synovial fluid. These proteins may serve as novel candidates for osteoarthritis biomarker development on a tissue-specific basis.

Non-invasive T1ρ mapping of the human cartilage response to loading and unloading.

OBJECTIVE: To define the physiological response to sequential loading and unloading in histologically intact human articular cartilage using serial T1ρ mapping, as T1ρ is considered to indicate the tissue's macromolecular content. METHOD: 18 macroscopically intact cartilage-bone samples were obtained from the central lateral femoral condyles of 18 patients undergoing total knee replacement. Serial T1ρ mapping was performed on a clinical 3.0-T MRI system using a modified prostate coil. Spin-lock multiple gradient-echo sequences prior to, during and after standardized indentation loading (displacement controlled, strain 20%) were used to obtain seven serial T1ρ maps: unloaded (δ0), quasi-statically loaded (indentation1-indentation3) and under subsequent relaxation (relaxation1-relaxation3). After manual segmentation, zonal and regional regions-of-interest were defined. ROI-specific relative changes were calculated and statistically assessed using paired t-tests. Histological (Mankin classification) and biomechanical (unconfined compression) evaluations served as references. RESULTS: All samples were histologically and biomechanically grossly intact (Mankin sum: 1.8 ± 1.2; Young's Modulus: 0.7 ± 0.4 MPa). Upon loading, T1ρ consistently increased throughout the entire sample thickness, primarily subpistonally (indentation1 [M ± SD]: 9.5 ± 7.8% [sub-pistonal area, SPA] vs 4.2 ± 5.8% [peri-pistonal area, PPA]; P < 0.001). T1ρ further increased with ongoing loading (indentation3: 14.1 ± 8.1 [SPA] vs 7.7 ± 5.9% [PPA]; P < 0.001). Even upon unloading (i.e., relaxation), T1ρ persistently increased in time. CONCLUSION: Serial T1ρ-mapping reveals distinct and complex zonal and regional changes in articular cartilage as a function of loading and unloading. Thereby, longitudinal adaptive processes in hyaline cartilage become evident, which may be used for the tissue's non-invasive functional characterization by T1ρ.

Full regeneration of segmental bone defects using porous titanium implants loaded with BMP-2 containing fibrin gels.

Regeneration of load-bearing segmental bone defects is a major challenge in trauma and orthopaedic surgery. The ideal bone graft substitute is a biomaterial that provides immediate mechanical stability, while stimulating bone regeneration to completely bridge defects over a short period. Therefore, selective laser melted porous titanium, designed and fine-tuned to tolerate full load-bearing, was filled with a physiologically concentrated fibrin gel loaded with bone morphogenetic protein-2 (BMP-2). This biomaterial was used to graft critical-sized segmental femoral bone defects in rats. As a control, porous titanium implants were either left empty or filled with a fibrin gels without BMP-2. We evaluated bone regeneration, bone quality and mechanical strength of grafted femora using in vivo and ex vivo µCT scanning, histology, and torsion testing. This biomaterial completely regenerated and bridged the critical-sized bone defects within eight weeks. After twelve weeks, femora were anatomically re-shaped and revealed open medullary cavities. More importantly, new bone was formed throughout the entire porous titanium implants and grafted femora regained more than their innate mechanical stability: torsional strength exceeded twice their original strength. In conclusion, combining porous titanium implants with a physiologically concentrated fibrin gels loaded with BMP-2 improved bone regeneration in load-bearing segmental defects. This material combination now awaits its evaluation in larger animal models to show its suitability for grafting load-bearing defects in trauma and orthopaedic surgery.

FK506 protects against articular cartilage collagenous extra-cellular matrix degradation.

OBJECTIVE: Osteoarthritis (OA) is a non-rheumatologic joint disease characterized by progressive degeneration of the cartilage extra-cellular matrix (ECM), enhanced subchondral bone remodeling, activation of synovial macrophages and osteophyte growth. Inhibition of calcineurin (Cn) activity through tacrolimus (FK506) in in vitro monolayer chondrocytes exerts positive effects on ECM marker expression. This study therefore investigated the effects of FK506 on anabolic and catabolic markers of osteoarthritic chondrocytes in 2D and 3D in vitro cultures, and its therapeutic effects in an in vivo rat model of OA. METHODS: Effects of high and low doses of FK506 on anabolic (QPCR/histochemistry) and catabolic (QPCR) markers were evaluated in vitro on isolated (2D) and ECM-embedded chondrocytes (explants, 3D pellets). Severe cartilage damage was induced unilaterally in rat knees using papain injections in combination with a moderate running protocol. Twenty rats were treated with FK506 orally and compared to twenty untreated controls. Subchondral cortical and trabecular bone changes (longitudinal microCT) and macrophage activation (SPECT/CT) were measured. Articular cartilage was analyzed ex vivo using contrast enhanced microCT and histology. RESULTS: FK506 treatment of osteoarthritic chondrocytes in vitro induced anabolic (mainly collagens) and reduced catabolic ECM marker expression. In line with this, FK506 treatment clearly protected ECM integrity in vivo by markedly decreasing subchondral sclerosis, less development of subchondral pores, depletion of synovial macrophage activation and lower osteophyte growth. CONCLUSION: FK506 protected cartilage matrix integrity in vitro and in vivo. Additionally, FK506 treatment in vivo reduced OA-like responses in different articular joint tissues and thereby makes Cn an interesting target for therapeutic intervention of OA.

Chondrogenically differentiated mesenchymal stromal cell pellets stimulate endochondral bone regeneration in critical-sized bone defects.

Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6 mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donor-dependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSC-based graft to repair large bone defects or atrophic non-unions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.

[New from old : relevant factors for fracture healing in aging bone]. / Aus alt mach neu : Relevante Faktoren bei der Frakturheilung im Alter.

BACKGROUND: Fracture healing is a complex biological process with specific temporal expression patterns. During this process new bone tissue is formed, which is similar to the original bone in quality and structure. This occurs in four phases: inflammation, formation of a soft tissue callus, formation of a bony callus and remodelling of the bony callus. This needs the precise orchestration of each cell type involved. OBJECTIVES: This article presents details of the fracture healing phases and the relevant factors. During the aging process there is an increase of reactive oxygen species and a change in expression pattern of growth factors that have a negative effect on the fracture healing process. METHODS: A selective review of the literature was carried out in PubMed concerning the influence of aging on fracture healing. CONCLUSION: The healing process is regulated by systemic and local factors. An understanding of these processes and the changes during aging is necessary in order to improve the knowledge of delayed or lack of fracture healing during aging to decide when an intervention is needed.

Physiomimetic biocompatibility evaluation of directly printed degradable porous iron implants using various cell types.

Additively manufactured (AM) degradable porous metallic biomaterials offer unique opportunities for satisfying the design requirements of an ideal bone substitute. Among the currently available biodegradable metals, iron has the highest elastic modulus, meaning that it would benefit the most from porous design. Given the successful preclinical applications of such biomaterials for the treatment of cardiovascular diseases, the moderate compatibility of AM porous iron with osteoblast-like cells, reported in earlier studies, has been surprising. This may be because, as opposed to static in vitro conditions, the biodegradation products of iron in vivo are transported away and excreted. To better mimic the in situ situations of biodegradable biomaterials after implantation, we compared the biodegradation behavior and cytocompatibility of AM porous iron under static conditions to the conditions with dynamic in situ-like fluid flow perfusion in a bioreactor. Furthermore, the compatibility of these scaffolds with four different cell types was evaluated to better understand the implications of these implants for the complex process of natural wound healing. These included endothelial cells, L929 fibroblasts, RAW264.7 macrophage-like cells, and osteoblastic MG-63 cells. The biodegradation rate of the scaffolds was significantly increased in the perfusion bioreactor as compared to static immersion. Under either condition, the compatibility with L929 cells was the best. Moreover, the compatibility with all the cell types was much enhanced under physiomimetic dynamic flow conditions as compared to static biodegradation. Our study highlights the importance of physiomimetic culture conditions and cell type selection when evaluating the cytocompatibility of degradable biomaterials in vitro. STATEMENT OF SIGNIFICANCE: Additively manufactured (AM) degradable porous metals offer unique opportunities for the treatment of large bony defects. Despite the successful preclinical applications of biodegradable iron in the cardiovascular field, the moderate compatibility of AM porous iron with osteoblast-like cells was reported. To better mimic the in vivo condition, we compared the biodegradation behavior and cytocompatibility of AM porous iron under static condition to dynamic perfusion. Furthermore, the compatibility of these scaffolds with various cell types was evaluated to better simulate the process of natural wound healing. Our study suggests that AM porous iron holds great promise for orthopedic applications, while also highlighting the importance of physio-mimetic culture conditions and cell type selection when evaluating the cytocompatibility of degradable biomaterials in vitro.

Additively manufactured biodegradable porous zinc.

Additively manufacturing (AM) opens up the possibility for biodegradable metals to possess uniquely combined characteristics that are desired for bone substitution, including bone-mimicking mechanical properties, topologically ordered porous structure, pore interconnectivity and biodegradability. Zinc is considered to be one of the promising biomaterials with respect to biodegradation rate and biocompatibility. However, no information regarding the biodegradability and biocompatibility of topologically ordered AM porous zinc is yet available. Here, we applied powder bed fusion to fabricate porous zinc with a topologically ordered diamond structure. An integrative study was conducted on the static and dynamic biodegradation behavior (in vitro, up to 4 weeks), evolution of mechanical properties with increasing immersion time, electrochemical performance, and biocompatibility of the AM porous zinc. The specimens lost 7.8% of their weight after 4 weeks of dynamic immersion in a revised simulated body fluid. The mechanisms of biodegradation were site-dependent and differed from the top of the specimens to the bottom. During the whole in vitro immersion time of 4 weeks, the elastic modulus values of the AM porous zinc (E = 700-1000 MPa) even increased and remained within the scope of those of cancellous bone. Indirect cytotoxicity revealed good cellular activity up to 72 h according to ISO 10,993-5 and -12. Live-dead staining confirmed good viability of MG-63 cells cultured on the surface of the AM porous zinc. These important findings could open up unprecedented opportunities for the development of multifunctional bone substituting materials that will enable reconstruction and regeneration of critical-size load-bearing bone defects. STATEMENT OF SIGNIFICANCE: No information regarding the biodegradability and biocompatibility of topologically ordered AM porous zinc is available. We applied selective laser melting to fabricate topologically ordered porous zinc and conducted a comprehensive study on the biodegradation behavior, electrochemical performance, time-dependent mechanical properties, and biocompatibility of the scaffolds. The specimens lost 7.8% of their weight after4 weeks dynamic biodegradation while their mechanical properties surprisingly increased after 4 weeks. Indirect cytotoxicity revealed good cellular activity up to 72 h. Intimate contact between MG-63 cells and the scaffolds was also observed. These important findings could open up unprecedented opportunities for the development of multifunctional bone substituting materials that mimic bone properties and enable full regeneration of critical-size load-bearing bony defects.

Additively manufactured functionally graded biodegradable porous zinc.

Topological design provides additively manufactured (AM) biodegradable porous metallic biomaterials with a unique opportunity to adjust their biodegradation behavior and mechanical properties, thereby satisfying the requirements for ideal bone substitutes. However, no information is available yet concerning the effect of topological design on the performance of AM porous zinc (Zn) that outperforms Mg and Fe in biodegradation behavior. Here, we studied one functionally graded and two uniform AM porous Zn designs with diamond unit cell. Cylindrical specimens were fabricated from pure Zn powder by using a powder bed fusion technique, followed by a comprehensive study on their static and dynamic biodegradation behaviors, mechanical properties, permeability, and biocompatibility. Topological design, indeed, affected the biodegradation behavior of the specimens, as evidenced by 150% variations in biodegradation rate between the three different designs. After in vitro dynamic immersion for 28 days, the AM porous Zn had weight losses of 7-12%, relying on the topological design. The degradation rates satisfied the desired biodegradation time of 1-2 years for bone substitution. The mechanical properties of the biodegraded specimens of all the groups maintained within the range of those of cancellous bone. As opposed to the trends observed for other biodegradable porous metals, after 28 days of in vitro biodegradation, the yield strengths of the specimens of all the groups (σy = 7-14 MPa) increased consistently, as compared to those of the as-built specimens (σy = 4-11 MPa). Moreover, AM porous Zn showed excellent biocompatibility, given that the cellular activities in none of the groups differed from the Ti controls for up to 72 h. Using topological design of AM porous Zn for controlling its mechanical properties and degradation behavior is thus clearly promising, thereby rendering flexibility to the material to meet a variety of clinical requirements.

Additively manufactured functionally graded biodegradable porous iron.

Additively manufactured (AM) functionally graded porous metallic biomaterials offer unique opportunities to satisfy the contradictory design requirements of an ideal bone substitute. However, no functionally graded porous structures have ever been 3D-printed from biodegradable metals, even though biodegradability is crucial both for full tissue regeneration and for the prevention of implant-associated infections in the long term. Here, we present the first ever report on AM functionally graded biodegradable porous metallic biomaterials. We made use of a diamond unit cell for the topological design of four different types of porous structures including two functionally graded structures and two reference uniform structures. Specimens were then fabricated from pure iron powder using selective laser melting (SLM), followed by experimental and computational analyses of their permeability, dynamic biodegradation behavior, mechanical properties, and cytocompatibility. It was found that the topological design with functional gradients controlled the fluid flow, mass transport properties and biodegradation behavior of the AM porous iron specimens, as up to 4-fold variations in permeability and up to 3-fold variations in biodegradation rate were observed for the different experimental groups. After 4 weeks of in vitro biodegradation, the AM porous scaffolds lost 5-16% of their weight. This falls into the desired range of biodegradation rates for bone substitution and confirms our hypothesis that topological design could indeed accelerate the biodegradation of otherwise slowly degrading metals, like iron. Even after 4 weeks of biodegradation, the mechanical properties of the specimens (i.e., E = 0.5-2.1 GPa, σy = 8-48 MPa) remained within the range of the values reported for trabecular bone. Design-dependent cell viability did not differ from gold standard controls for up to 48 h. This study clearly shows the great potential of AM functionally graded porous iron as a bone substituting material. Moreover, we demonstrate that complex topological design permits the control of mechanical properties, degradation behavior of AM porous metallic biomaterials. STATEMENT OF SIGNIFICANCE: No functionally graded porous structures have ever been 3D-printed from biodegradable metals, even though biodegradability is crucial both for full tissue regeneration and for the prevention of implant-associated infections in the long term. Here, we present the first report on 3D-printed functionally graded biodegradable porous metallic biomaterials. Our results suggest that topological design in general, and functional gradients in particular can be used as an important tool for adjusting the biodegradation behavior of AM porous metallic biomaterials. The biodegradation rate and mass transport properties of AM porous iron can be increased while maintaining the bone-mimicking mechanical properties of these biomaterials. The observations reported here underline the importance of proper topological design in the development of AM porous biodegradable metals.

Glucosamine increases hyaluronic acid production in human osteoarthritic synovium explants.

BACKGROUND: Glucosamine (GlcN) used by patients with osteoarthritis was demonstrated to reduce pain, but the working mechanism is still not clear. Viscosupplementation with hyaluronic acid (HA) is also described to reduce pain in osteoarthritis. The synthesis of HA requires GlcN as one of its main building blocks. We therefore hypothesized that addition of GlcN might increase HA production by synovium tissue. METHODS: Human osteoarthritic synovium explants were obtained at total knee surgery and pre-cultured for 1 day. The experimental conditions consisted of a 2 days continuation of the culture with addition of N-Acetyl-glucosamine (GlcN-Ac; 5 mM), glucosamine-hydrochloride (GlcN-HCl; 0.5 and 5 mM), glucose (Gluc; 0.5 and 5 mM). Hereafter HA production was measured in culture medium supernatant using an enzyme-linked binding protein assay. Real time RT-PCR was performed for hyaluronic acid synthase (HAS) 1, 2 and 3 on RNA isolated from the explants. RESULTS: 0.5 mM and 5 mM GlcN-HCl significantly increased HA production compared to control (approximately 2 - 4-fold), whereas GlcN-Ac had no significant effect. Addition of 5 mM Gluc also increased HA production (approximately 2-fold), but 0.5 mM Gluc did not. Gene expression of the HA forming enzymes HAS 1, 2 and 3 was not altered by the addition of GlcN or Gluc. CONCLUSION: Our data suggest that exogenous GlcN can increase HA production by synovium tissue and is more effective at lower concentrations than Gluc. This might indicate that GlcN exerts its potential analgesic properties through stimulation of synovial HA production.

Additively manufactured biodegradable porous magnesium.

An ideal bone substituting material should be bone-mimicking in terms of mechanical properties, present a precisely controlled and fully interconnected porous structure, and degrade in the human body to allow for full regeneration of large bony defects. However, simultaneously satisfying all these three requirements has so far been highly challenging. Here we present topologically ordered porous magnesium (WE43) scaffolds based on the diamond unit cell that were fabricated by selective laser melting (SLM) and satisfy all the requirements. We studied the in vitro biodegradation behavior (up to 4 weeks), mechanical properties and biocompatibility of the developed scaffolds. The mechanical properties of the AM porous WE43 (E = 700-800 MPa) scaffolds were found to fall into the range of the values reported for trabecular bone even after 4 weeks of biodegradation. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), electrochemical tests and µCT revealed a unique biodegradation mechanism that started with uniform corrosion, followed by localized corrosion, particularly in the center of the scaffolds. Biocompatibility tests performed up to 72 h showed level 0 cytotoxicity (according to ISO 10993-5 and -12), except for one time point (i.e., 24 h). Intimate contact between cells (MG-63) and the scaffolds was also observed in SEM images. The study shows for the first time that AM of porous Mg may provide distinct possibilities to adjust biodegradation profile through topological design and open up unprecedented opportunities to develop multifunctional bone substituting materials that mimic bone properties and enable full regeneration of critical-size load-bearing bony defects. STATEMENT OF SIGNIFICANCE: The ideal biomaterials for bone tissue regeneration should be bone-mimicking in terms of mechanical properties, present a fully interconnected porous structure, and exhibit a specific biodegradation behavior to enable full regeneration of bony defects. Recent advances in additive manufacturing have resulted in biomaterials that satisfy the first two requirements but simultaneously satisfying the third requirement has proven challenging so far. Here we present additively manufactured porous magnesium structures that have the potential to satisfy all above-mentioned requirements. Even after 4 weeks of biodegradation, the mechanical properties of the porous structures were found to be within those reported for native bone. Moreover, our comprehensive electrochemical, mechanical, topological, and biological study revealed a unique biodegradation behavior and the limited cytotoxicity of the developed biomaterials.

Additively manufactured biodegradable porous iron.

Additively manufactured (AM) topologically ordered porous metallic biomaterials with the proper biodegradation profile offer a unique combination of properties ideal for bone regeneration. These include a fully interconnected porous structure, bone-mimicking mechanical properties, and the possibility of fully regenerating bony defects. Most of such biomaterials are, however, based on magnesium and, thus, degrade too fast. Here, we present the first report on topologically ordered porous iron made by Direct Metal Printing (DMP). The topological design was based on a repetitive diamond unit cell. We conducted a comprehensive study on the in vitro biodegradation behavior (up to 28 days), electrochemical performance, time-dependent mechanical properties, and biocompatibility of the scaffolds. The mechanical properties of AM porous iron (E = 1600-1800 MPa) were still within the range of the values reported for trabecular bone after 28 days of biodegradation. Electrochemical tests showed up to ≈12 times higher rates of biodegradation for AM porous iron as compared to that of cold-rolled (CR) iron, while only 3.1% of weight loss was measured after 4 weeks of immersion tests. The biodegradation mechanisms were found to be topology-dependent and different between the periphery and central parts of the scaffolds. While direct contact between MG-63 cells and scaffolds revealed substantial and almost instant cytotoxicity in static cell culture, as compared to Ti-6Al-4V, the cytocompatibility according to ISO 10993 was reasonable in in vitro assays for up to 72 h. This study shows how DMP could be used to increase the surface area and decrease the grain sizes of topologically ordered porous metallic biomaterials made from metals that are usually considered to degrade too slowly (e.g., iron), opening up many new opportunities for the development of biodegradable metallic biomaterials. STATEMENT OF SIGNIFICANCE: Biodegradation in general and proper biodegradation profile in particular are perhaps the most important requirements that additively manufactured (AM) topologically ordered porous metallic biomaterials should offer in order to become the ideal biomaterial for bone regeneration. Currently, most biodegradable metallic biomaterials are based on magnesium, which degrade fast with gas generation. Here, we present the first report on topologically ordered porous iron made by Direct Metal Printing (DMP). We also conducted a comprehensive study on the biodegradation behavior, electrochemical performance, biocompatibility, and the time evolution of the mechanical properties of the implants. We show that these implants possess bone-mimicking mechanical properties, accelerated degradation rate, and reasonable cytocompatibility, opening up many new opportunities for the development of iron-based biodegradable materials.

Effect of pretransplant preconditioning by whole body hyperthermia on islet graft survival.

Previous observations in heat-shocked pig islets revealed the ambivalent character of the stress response simultaneously inducing processes of protection and apoptosis. To clarify whether the proapoptotic character of the stress response is reduced in heat-exposed islets still embedded in their native environment, hyperthermia was performed in the present study either as whole body hyperthermia (WBH) prior to pancreas resection or as in vitro heat shock (HS) after isolation. HS (42 degrees C/45 min) was induced in donors 12 h before isolation (WBH, n = 32) or in freshly isolated islets prior to 12 h of culture at 37 degrees C (in vitro HS, n = 25). Islets continuously incubated at 37 degrees C served as controls (n = 34). Proinflammatory treatment was performed with H2O2, DETA-NO, or a combination of IL-1beta, TNF-alpha, and IFN-gamma. Quality assessment included islet yield, viability staining, static glucose incubation, and nude mouse transplantation. WBH was significantly less effective than in vitro HS to induce HSP70 overexpression and to increase islet resistance against inflammatory mediators. Although characterized by an unaltered Bax to Bcl-2 ratio, islets subjected to WBH partially failed to restore sustained normoglycemia in diabetic nude mice. The inflammatory response observed in the pancreas of WBH-treated rats was associated with significantly reduced viability that seems to have a higher predictive value for posttransplant outcome compared to islet in vitro function or mitochondrial activity. In contrast, in vitro HS significantly decreased transcript levels of Bcl-2, but did not affect posttransplant function compared to sham-treated islets. These findings suggest that WBH is primarily associated with increased necrosis as a secondary tissue type-specific effect of pancreas damage while in vitro HS mainly induces apoptosis.

Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a.

Recent studies demonstrate widespread expression of ghrelin among tissues and have uncovered its pleiotropic nature. We have examined gene expression of ghrelin and its two receptor splice variants, growth hormone secretagogue receptors (GHS-R) 1a and 1b, in human bone biopsies and in the human pre-osteoblastic SV-HFO cell line during differentiation. Additionally, we examined proliferative effects of ghrelin and unacylated ghrelin (UAG) in differentiating and non-differentiating cells. We detected GHS-R1b mRNA in human bone and osteoblasts but not ghrelin's cognate receptor GHS-R1a, using two different real-time PCR assays and both total RNA and mRNA. In osteoblasts GHS-R1b mRNA expression remained low during the first 14 days of culture, but increased 300% in differentiating cells by day 21. Both human bone biopsies and osteoblasts expressed ghrelin mRNA, and osteoblasts were found to secrete ghrelin. Overall, ghrelin gene expression was greater in differentiating than non-differentiating osteoblasts, but was not increased during culture in either group. Ghrelin and UAG induced thymidine uptake dose-dependently, peaking at 1 and 10 nM respectively, at day 6 of culture in both non-differentiating and differentiating osteoblasts. The proliferative response to ghrelin and UAG declined with culture time and state of differentiation. The proliferative effects of ghrelin and UAG were suppressed by inhibitors of extracellular-signal-regulated kinase (ERK) and phosphoinositide-3 kinase, and both peptides rapidly induced ERK phosphorylation. Overall, our data suggest new roles for ghrelin and UAG in modulating human osteoblast proliferation via a novel signal transduction pathway.

Demonstration of insulin degradation by thiol-protein disulfide oxidoreductase (glutathione-insulin transhydrogenase) and proteinases of pancreatic islets.

Homogenate preparations of pancreatic islets have been found to degrade insulin by cleavage of the interchain disulfide bonds, followed by proteolysis of the resulting A and B chains. A proteolytic system of the pancreatic islets splitting not only 125I-labeled insulin A chain but also 125I-labeled glucagon at pH 7.0, was shown to be activated by glutathione and inhibited by EDTA. The results suggest that pancreatic islets contain both the thiol-protein disulfide oxidoreductase (glutathione : protein-disulfide oxidoreductase, EC 1.8.4.2) and the A and B chain-degrading enzyme(s). The effects of EDTA argue against the implication of cathepsins in insulin breakdown under the experimental conditions employed.

Endotoxin impairs the engraftment of rat islets transplanted beneath the kidney capsule of C57BL/6-mice.

The primary objective of this investigation was to determine the effect of endotoxin on islet xenograft survival within the first three days after transplantation. Pancreatic islets from Lewis rats were prepared under endotoxin-free conditions with Liberase (Boehringer) and purified by centrifugation on endotoxin-free Ficoll/Histopaque. After overnight incubation, with or without 10 microg/ml endotoxin, the islets were transplanted beneath the kidney capsule of normoglycemic C57Bl/6-mice. Three days later, kidneys were removed and their insulin content were measured. We could demonstrate significant differences (P<0.01) in insulin recovery between lipopolysaccharide-free and lipopolysaccharide-containing grafts. In case of endotoxin contaminated islets, we found only 13+/-2% (n=9) of the original insulin content, in contrast to 53+/-7% (n=9) when endotoxin-free islets where grafted. In experiments with islets isolated by use of conventional (lipopolysaccharide-containing) collagenase, and then cultured in endotoxin-free medium, insulin recovery three days after transplantation was 36+/-1% (n=13).

Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll.

Endotoxin-induced early inflammatory reactions may inhibit the function and survival of isolated cells or cell aggregates after transplantation. By the chromogenic Limulus amebocyte lysate assay we found rather high but variable endotoxin concentrations in the chemicals used for islet isolation, i.e. collagenase and Ficoll. Liberase, a special collagenase preparation from Boehringer, was nearly endotoxin-free. Correlating to the endotoxin content, collagenase and Ficoll had the capacity to induce interleukin-1beta release from human peripheral blood mononuclear cells. Because collagenase and density gradient media are needed in most cell isolation procedures from solid organs, each lot of these chemicals should be tested for endotoxin contamination. In pancreatic islet transplantation, the use of endotoxin-free chemicals may diminish early local inflammatory reactions at the graft site and thereby reduce the number of islets needed for successful islet transplantation.

Oxygen radical production in human mononuclear blood cells is not suppressed by drugs used in clinical islet transplantation.

Inflammatory islet damage mediated by cytokines and oxygen radicals may limit the success of clinical islet transplantation for treatment of insulin-dependent diabetes mellitus. In this study, we investigated whether drugs such as currently used in islet-transplanted patients inhibit the release of IL-1beta, TNFalpha, and superoxide from mononuclear blood cells in vitro. Methylprednisolone (10 microg/ml) inhibited the release of IL-1beta and TNFalpha, but had no effect on superoxide generation. Both pentoxifylline (66 microg/ml) and cyclosporin A (300 ng/ml) slightly inhibited TNFalpha release without affecting IL-1beta or superoxide generation. Nicotinamide (0.25 mM) did not interfere with the generation TNFalpha or superoxide and only slightly inhibited IL-1beta production. A combination of methylprednisolone, pentoxifylline, cyclosporin A, and nicotinamide (concentrations for each substance as described above) inhibited TNFalpha generation by 74+/-6% (mean value+/-SEM, mononuclear blood cells from seven diabetic patients) without affecting IL-1beta or superoxide generation. These data show that standard immunosuppressive therapy in islet transplanted patients may partially inhibit cytokine release but does not affect the generation of potentially islet-toxic superoxide from mononuclear cells.

Cytokine mRNA expression in peripheral blood cells of immunosuppressed human islet transplant recipients.

The macrophage derived cytokines interleukin-beta (IL-1beta), and tumor necrosis factor alpha (TNFalpha), and the T-cell derived cytokine interferon gamma (IFNgamma) have been implicated to play an important role in early attack on islet cells during human islet transplantation (ITx). Therefore, the aim of this study was to investigate the influence of the current immunosuppressive induction therapy in clinical islet transplantation on mRNA expression of these cytokines in blood cells, compared to lipopolysaccharide (LPS) induced cytokine release in vitro and to plasma levels. The cytokine release correlated to lymphocyte counts and significantly decreased after ATG, and partially recovered 2 weeks after ITx. Unexpectedly, there was no correlation between mRNA expression for IL-1beta in total blood and the number of lymphocytes and monocytes remaining after anti thymocyte globulin (ATG)-therapy. Even when the blood was nearly totally depleted from mononuclear cells, high amounts of IL-1beta mRNA could be detected. However, IL-1beta secretion could not be stimulated in vitro. Our results show that application of ATG during ITx might contribute to graft survival during the early posttransplant period by suppression of the synthesis of monocyte derived cytokines IL-1beta and TNFalpha.