An EAACI review: Go green in health care and research. Practical suggestions for sustainability in clinical practice, laboratories, and scientific meetings.

Health care professionals (HCPs) and researchers in the health care sector dedicate their professional life to maintaining and optimizing the health of their patients. To achieve this, significant amounts of resources are used and currently it is estimated that the health care sector contributes to more than 4% of net greenhouse gas (GHG) emissions. GHG emissions adversely impact planetary health and consequently human health, as the two are intricately linked. There are many factors of health care that contribute to these emissions. Hospitals and research labs also use high amounts of consumables which require large amounts of raw materials and energy to produce. They are further responsible for polluting the environment via disposal of plastics, drug products, and other chemicals. To maintain and develop state-of-the-art best practices and treatments, medical experts exchange and update their knowledge on methods and technologies in the respective fields at highly specialized scientific meetings. These meetings necessitate thousands of attendants traveling around the globe. Therefore, while the goal of HCPs is to care for the individual, current practices have an enormous (indirect) impact on the health of the patients by their negative environmental impacts. There is an urgent need for HCPs and researchers to mitigate these detrimental effects. The installation of a sustainability-manager at health care facilities and research organizations to implement sustainable practices while still providing quality health care is desirable. Increased use of telemedicine, virtual/hybrid conferences and green chemistry have recently been observed. The benefits of these practices need to be evaluated and implemented as appropriate. With this manuscript, we aim to increase the awareness about the negative impacts of the health care system (including health care research) on planetary and human health. We suggest some easy and highly impactful steps and encourage health care professionals and research scientists of all hierarchical levels to immediately implement them in their professional as well as private life to counteract the health care sector's detrimental effects on the environment.

Increased bone formation in a rabbit long-bone defect model after single local and single systemic application of erythropoietin.

Background and purpose - Delayed bone healing with non-union is a common problem. Further options to increase bone healing together with surgery are needed. We therefore evaluated a 1-dose single application of erythropoietin (EPO), applied either locally to the defect or systemically during surgery, in a critical-size rabbit long-bone defect. Material and methods - 19 New Zealand White rabbits received a 15-mm defect in the radius diaphysis. An absorbable gelatin sponge was soaked with saline (control group and systemic treatment group) or EPO (local treatment group) and implanted into the gap. The systemic treatment group received EPO subcutaneously. In vivo micro-CT analysis was performed 4, 8, and 12 weeks postoperatively. Vascularization was evaluated histologically. Results - Semiquantitative histomorphometric and radiological evaluation showed increased bone formation (2.3- to 2.5-fold) in both treatment groups after 12 weeks compared to the controls. Quantitative determination of bone volume and tissue volume showed superior bone healing after EPO treatment at all follow-up time points, with the highest values after 12 weeks in locally treated animals (3.0- to 3.4-fold). More vascularization was found in both EPO treatment groups. Interpretation - Initial single dosing with EPO was sufficient to increase bone healing substantially after 12 weeks of follow-up. Local application inside the defect was most effective, and it can be administered directly during surgery. Apart from effects on ossification, systemic and local EPO treatment leads to increased callus vascularization.

Mesenchymal stromal cell implantation for stimulation of long bone healing aggravates Staphylococcus aureus induced osteomyelitis.

Large bone defects requiring long-term osteosynthetic stabilization or repeated surgeries show a considerable rate of infection. Mesenchymal stromal cells (MSCs) have been successfully used to enhance bone regeneration, but their powerful immunomodulatory effects may impose an enhanced risk for osteomyelitis development. In order to unravel whether implantation of MSCs aggravates a simultaneous bone infection, a hydrogel-supported osteomyelitis ostectomy model was developed in which rats received a femoral bone defect with rigid plate-fixation. After fibrin-assisted transfer of Staphylococcus aureus (SA), effects of MSC implantation on osteomyelitis development were quantified over 3-4 weeks. All SA-infected animals developed an acute local osteomyelitis with significantly increased blood neutrophil count, abscess formation and bone destruction. MSC-treatment of infected defects aggravated osteomyelitis according to a significantly elevated osteomyelitis score and enhanced distal bone loss with spongy alteration of cortical bone architecture. Increased attraction of macrophages, osteoclasts and regulation of pro- and anti-inflammatory mediators were potential MSC actions. Overall trophic actions of MSCs implanted into non-sterile bone defects may enhance an infection and/or exacerbate osteomyelitis. Studies on antibiotic carrier augmentation or antibiotic treatment are warranted to decide whether MSC implantation is a safe and promising therapy for orthopedic implant-stabilized bone defects at high risk for development of infection.

Superior angiogenic potential of GDF-5 and GDF-5(V453/V456) compared with BMP-2 in a rabbit long-bone defect model.

BACKGROUND: The clinical application of bone morphogenetic proteins such as BMP-2 and GDF-5 (growth and differentiation factor-5) may improve the outcome of bone defect repair. In addition to the osteoinductivity of BMPs, their angiogenic potential is important as an adequate blood supply is a prerequisite for bone-healing. We used a rabbit long-bone defect model to investigate whether angiogenicity and osteogenicity were correlated features of a BMP molecule by comparing the induction of blood vessel and bone formation by BMP-2, GDF-5, and a previously created swap mutant GDF-5V453/V456 (BB-1) with elevated BMP receptor-IA binding. METHODS: Microcomputed tomography and immunohistochemistry were used to assess early bone formation and neovascularization in 15-mm (critical-sized) rabbit radius defects treated with a growth factor-loaded collagen carrier. RESULTS: Blood vessel volume and surface area on days 7 and 14 after surgery were significantly greater in defects treated with GDF-5 and with BB-1 compared with controls (p < 0.05); BMP-2 enhanced vascularization on day 14 (p < 0.05). Cumulative data including both time points reflected increased vessel volume, intersection surface area, and number of vessels after treatment with GDF-5 and BB-1 compared with BMP-2 (p < 0.05), corresponding to the histology results. Each of the growth factors resulted in enhanced bone formation compared with controls on day 14 (p < 0.01), with BB-1 resulting in significantly more bone compared with GDF-5 as indicated by bone volume and surface area (p = 0.006). CONCLUSIONS: Both GDF-5 and BB-1 had high angiogenicity, and BB-1 outperformed GDF-5 with respect to osteogenicity. Strong induction of bone formation by BMP-2 and BB-1 was thus associated with BMP receptor-IA-dependent signaling, whereas the vascularization outcome was not. CLINICAL RELEVANCE: Although both BMP-2 and the GDF-5 variant BB-1 are good inducers of bone formation, BB-1 is especially promising for long-bone healing if high angiogenicity is desired along with high osteogenicity to promote recreation of optimal bone architecture.

Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis.

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT-analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA-pathology. Our data suggest that MC1-signaling protects against cartilage degradation and subchondral bone sclerosis in OA indicating a beneficial role of the POMC system in joint pathophysiology.

Identification of novel SHOX target genes in the developing limb using a transgenic mouse model.

Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development.

Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF-5 and BMP-2.

Non healing bone defects remain a worldwide health problem and still only few osteoinductive growth factors are available for clinical use in bone regeneration. By introducing BMP-2 residues into growth and differentiation factor (GDF)-5 we recently produced a mutant GDF-5 protein BB-1 which enhanced heterotopic bone formation in mice. Designed to combine positive features of GDF-5 and BMP-2, we suspected that this new growth factor variant may improve long bone healing compared to the parent molecules and intended to unravel functional mechanisms behind its action. BB-1 acquired an increased binding affinity to the BMP-IA receptor, mediated enhanced osteogenic induction of human mesenchymal stem cells versus GDF-5 and higher VEGF secretion than BMP-2 in vitro. Rabbit radius defects treated with a BB-1-coated collagen carrier healed earlier and with increased bone volume compared to BMP-2 and GDF-5 according to in vivo micro-CT follow-up. While BMP-2 callus often remained spongy, BB-1 supported earlier corticalis and marrow cavity formation, showing no pseudojoint persistence like with GDF-5. Thus, by combining positive angiogenic and osteogenic features of GDF-5 and BMP-2, only BB-1 restored a natural bone architecture within 12 weeks, rendering this promising growth factor variant especially promising for long bone regeneration.

Alginate encapsulated human mesenchymal stem cells suppress syngeneic glioma growth in the immunocompetent rat.

Human mesenchymal stem cells (MSC) are promising candidates for cell therapy of neurological diseases. However, co-transplantation of MSC with tumour cell lines has been reported to promote tumour growth. In this study, we co-transplant glioma cells together with alginate-encapsulated MSC. Immunocompetent BD-IX rats were inoculated with syngeneic BT4Ca glioma cells. Encapsulated unmodified MSC, endostatin producing (endoMSC) or cell-free alginate capsules were stereotactically implanted into the tumour bed. After 12 days, tumour volumes were significantly diminished in the MSC-treated group. The decrease in tumour volume found with endoMSC was statistically not significant, despite significantly reduced tumour vascularization. We conclude that, under syngeneic conditions in the immunocompetent animal, (1) the intracranial, orthotopic co-transplantation of MSC with glioma cells leads to a suppression in tumour growth and (2) the tumour can escape the antiangiogenic treatment with endostatin. Our finding may facilitate the clinical translation of encapsulated cell therapy.

Bone cement penetration pattern and primary stability testing in keeled and pegged glenoid components.

BACKGROUND: It has been proposed that bone mineral density has an influence on cement penetration in hip and knee arthroplasty. The hypotheses of this study were that: 1) there is a negative correlation between bone mineral density (BMD) and cement penetration in cemented glenoid components; and 2) that implant design has an influence on cement penetration into the glenoid bone. METHODS: BMD of 10 pairs of fresh frozen scapulas was measured. Micro-computed tomography (micro-CT) scans in 3 different sections were analyzed after implantation of keeled and pegged glenoid components using a 3(rd)-generation cementing technique with a vacuum mixing system. Cement penetration was analyzed and correlated with BMD. Pull-out strength testing was performed to analyze primary stability. RESULTS: The overall peak BMD was 0.6 [g/cm(2)] (range, 0.33-0.98). A strong negative correlation between BMD and mean cement penetration was found for the peg (R(2) = -.83; P < .003) and for the keel group (R(2) = -.81; P < .005). Mean cement penetration was 78.4 mm(2) (range, 60.6-94.2) in the keel and 113.9 mm(2) (range, 78.2-143.4) in the peg group (P < .0001). In all cases, the components were pulled out of the cement mantle, whereas the bone-cement interfaces remained intact. The mean pull-out strength was 1093N (764-1343N) for keeled and 884N (650-1264N) for pegged components (P < .05). CONCLUSION: A modern cementing technique, leading to a deep bonding between bone and cement, is crucial to prevent loosening of glenoid components. The findings of this study might help us to better understand the results of follow-up studies of cemented glenoid implants. Our results could be helpful for the choice of implants in patients with poor bone quality like osteoporosis or rheumatoid arthritis.

Chondrogenic pre-induction of human mesenchymal stem cells on beta-TCP: enhanced bone quality by endochondral heterotopic bone formation.

New techniques to heal bone defects include the combination of bone substitute materials with mesenchymal stem cells (MSC). To find solutions not hampered by low material resorbability or high donor variability of human MSC, the potency of such composites is usually evaluated by heterotopic bone formation assays in immunocompromised animals. The aim of this study was to investigate whether resorbable phase-pure beta-tricalcium-phosphate (beta-TCP) could support heterotopic bone formation by MSC comparable to partially resorbable hydroxyapatite/tricalcium-phosphate (HA/TCP). Furthermore, in light of disappointing results with osteogenic in vitro priming of MSC, we tested whether chondrogenic pre-induction of constructs may allow for enhanced bone formation by triggering the endochondral pathway. beta-TCP granules of three different sizes and HA/TCP were seeded with MSC and transplanted subcutaneously into immunocompromised mice either immediately or after a chondrogenic pre-induction for 6 weeks. After 8 weeks, explants were analysed by histology. beta-TCP seeded with unprimed MSC revealed intramembranous bone formation without haematopoietic marrow with 3.8-fold more bone formed with granules smaller than 0.7 mm than with 0.7-1.4mm particles (p< or =0.018). Chondrogenic pre-induction of beta-TCP/MSC composites resulted in collagen type II and proteoglycan-rich cartilage-like tissue which, after transplantation, underwent endochondral ossification, yielding ectopic bone produced by human cells while haematopoietic marrow was derived from the mouse. Transdifferentiation of MSC-derived chondrocytes to osteoblasts or direct osteogenesis of cartilage-resident MSC is postulated to explain the human origin of new bone. In conclusion, beta-TCP was significantly more osteo-permissive (p=0.004) than HA/TCP for human MSC, and chondrogenic priming of beta-TCP/MSC represented a superior approach capable of supporting full bone formation, including marrow organization.