Det pågår omfattande studier, framför allt i USA och Storbritannien, som undersöker möjligheterna att införa screening med hjälp av blodprover för tidig upptäckt av ett flertal cancerformer, så kallade MCED (Mulitple Cancer Early Detection). Ännu har inget faktiskt screeningsprogram med MCED-screening initierats i världen. I Sverige ställs idag höga krav på evidens från studier för att ett screeningprogram ska kunna införas. Det pågår studier som adresserar frågan om screening, men än så länge saknas mycket information. Med utgångspunkt i denna rapport kan därför inte en initiering av MCEDscreening i Sverige rekommenderas i dagsläget. För att vara i framkant bör Sverige dock följa området nogsamt. Grail är ett väletablerat företag som i augusti 2021 blev uppköpt av Illumina, ett företag som 2020 rapporterade en vinst på 2,34 miljarder USD. Företaget har stora ambitioner för framtida screeningprogram och kan bedömas ha de resurser som krävs att genomföra essentiella studier. De har säkrade medel och genomför projekt för att utveckla sin produkt för att fungera i en screeningsituation. Storbritanniens stora pågående studie ”NHS-Galleri Clinical Trial” beräknas publicera nya resultat under 2023. Om denna studie faller ut väl planeras en utvidgning av studien som ska omfatta en miljon personer. Denna större studie beräknas kunna avslutas 2030. Data att följa i nämnda ovanstående studie är bland annat: - Hur stor population ännu icke diagnostiserade utgör, i empiriska siffror - Utvecklingen av testmetoden gällande sensibilitet och sensitivitet - Patientnyttan, t.ex. ökar överlevnaden mot dagens siffror - Risken för överdiagnostik - Kostnader och logistik som kommer belasta sjukvården En problematik med MCED-screening är att testernas specificitet och sensitivitet behöver vara tillräckligt tillförlitliga för att det inte skall belasta snarare hjälpa samhället. Det är osannolikt att dessa värden kommer att vara 100-procentiga, men hur bra dessa värden bör vara innan man inför en screening är öppet för diskussion, däri ligger problematiken. I rapporten förklaras dessa begrepp i mer detalj. Det bör nämnas att Grail redan har kunnat presentera en ökad prestanda utifrån preliminära resultat ifrån pågående studier som presenterats i en så kallad sub-studie, där sensitivitet, sensibilitet och ”Tissue of Origin” (TOO) har förbättrats ytterligare gällande tolv olika cancerformer. Beräkningar i vår rapport har nyttjat dessa värden för att söka ge en bild över vad som kan förväntas vid ett införande av en Grail-MCED-screening i Sverige. För de tolv cancerformerna kan beräkningarna sammanfattas enligt; - Under förutsättning att det finns adekvat behandling att erbjuda vid tidigare diagnostik, kommer det ske en markant förskjutning av i vilket stadie som diagnostiserade individer detekteras och förhoppningsvis behandlas. Idag behandlas cirka 40 -45% av de cancerdiagnostiserade i stadie III/IV, att jämföras mot de beräknade 6% vid införande av årlig screening. - Ökningen av antalet individer som skulle överleva sin cancer uppskattas till mellan 3 - 5 000 individer, utöver de cirka 9 000 som överlever med hjälp av dagens diagnostik/behandling. - 25 – 35% kostnadsreducering både vad gäller direkta kostnader och samhällskostnader, extrapolerat från IHE rapport (1). Vi har dock inte tagit hänsyn till de kostnadsökningar ett införande av en MCED-screening skulle innebära. Det reella värdet av rapportens beräkningar bör vägas mot de empiriska data som kommer produceras av NHS studie av Grails metod. Starka avvikelser (”positiva/negativa”) från empiriska data kan då ligga till grund för om och i så fall hur pass snabbt man vill driva införande av MCED screening i Sverige. Rapportens sammantagna bedömning är att de av Socialstyrelsen uppsatta kriterier för införande av en screening, inte är uppfyllda i dagsläget. Men vi har även försökt visualisera vad som skulle komma att ske med dagens diagnostiserade cancerpatientgrupper om en MCED-screening införs i ålderspopulationen 40 – 80 år (96% av dagens diagnostiserade), på de tolv cancerformer som metoden visar bäst sensibilitet/sensitivitet mot. Dessa tolv cancerformer utgör 36% av dagens diagnostiserade patienter. Trots att ett införande av screening inte är att rekommendera i dagsläget, finns det alternativ att ta ställning till; - Endast avvakta utredningarna i Storbritannien (till 2023, eller till 2030). - Avvakta utredningarna men initiera samtal med beslutsfattare som styr över de ekonomiska medel som kan bistå med satsningar på större Grailtester i Sverige - Avvakta utredningarna, men initiera samtal med både NHS och Grail i syfte att behålla momentum i detta första initiativ till utredning. Författarnas uppfattning är att Grails teknik är signifikant och kommer med stor sannolikhet bedömas som tillämpbar för MCED-screening i framtiden.

Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication, and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus, and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation, and prepare for future in-vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulfate (protocol 1) or 2% sodium deoxycholate (protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support fetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (protocol 2) was the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for two weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in-vivo sheep uterus bioengineering experiments.

Scaffold characteristics are decisive for repopulating the acellular tissue with cells. A method to produce such a scaffold from intact organ requires a customized decellularization protocol. Here, we have decellularized whole, intact porcine hearts by serial perfusion and agitation of hypotonic solution, an ionic detergent (4% sodium deoxycholate), and a nonionic detergent (1% Triton X-100). The resultant matrix was characterized for its degree of decellularization, morphological and functional integrity. The protocol used resulted in extensive decellularization of the cardiac tissue, but the cytoskeletal elements (contractile apparatus) of cardiomyocytes remained largely unaffected by the procedure although their membranous organelles were completely absent. Further, several residual angiogenic growth factors were found to be present in the decellularized tissue.

Vascular diseases are increasing health problems affecting >25 million individuals in westernized societies. Such patients could benefit fromtransplantation of tissue-engineered vascular grafts using autologous cells. One challenge that has limited this development is the need for cell isolation, and risks associated with ex vivo expanded stem cells. Herewe demonstrate a novel approach to generate transplantable vascular grafts using decellularized allogeneic vascular scaffolds, repopulatedwith peripheralwhole blood (PWB) in vitro in a bioreactor. Circulating, VEGFR-2+/CD45+ and a smaller fraction of VEGFR-2+/CD14+ cells contributed to repopulation of the graft. SEMmicrographs showed flat cells on the luminal surface of the grafts consistentwith endothelial cells. For clinical validation, two autologous PWBtissue-engineered vein conduits were prepared and successfully used for bypass procedures in two pediatric patients. These results provide a proof of principle for the generation of transplantable vascular grafts using a simple autologous blood sample, making it clinically feasible globally.

Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H2O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH2O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.

We aimed to produce an acellular human tissue scaffold with a view to test the possibility of recellularization with bone marrow stem cells to produce a tissue-engineered small intestine (TESI). Human small-bowel specimens (n = 5) were obtained from cadaveric organ donors and treated sequentially with 6% dimethyl sulfoxide in hypotonic buffer, 1% Triton X-100, and DNase. Each small intestine (SI) piece (6 cm) was recellularized with EPCAM+ and CD133+ allogeneic bone marrow stem cells. Histological and molecular analysis demonstrated that after decellularization, all cellular components and nuclear material were removed. Our analysis also showed that the decellularized human SI tissue retained its histoarchitecture with intact villi and major structural proteins. Protein films of common extracellular matrix constituents (collagen I, laminin, and fibronectin) were found in abundance. Furthermore, several residual angiogenic factors were found in the decellularized SI. Following recellularization, we found viable mucin-positive goblet cells, CK18+ epithelial cells in villi adjacent to a muscularis mucosa with α-actin+ smooth muscle cells, and a high repopulation of blood vessels with CD31+ endothelial cells. Our results show that in the future, such a TESI would be ideal for clinical purposes, because it can be derived from the recipient's own immunocompatible bone marrow cells, thus avoiding the use of immunosuppression.

OBJECTIVES: Many patients in need of bypass surgery lack graft material and current synthetic alternatives have poor performance. A 4 mm vascular graft composed of bacterial cellulose (BC) was developed and tested in pilot study in a large animal model. DESIGN: BC is a biopolymer made by the bacteria acetobacter xylinum. BC grafts (n = 16) with 4 cm length and 4 mm internal diameter were implanted bilaterally in the carotid arteries of eight sheep. No long-term antithrombotic therapy was administered. Patency was assessed with ultrasound. Histology, immunohistochemistry, and electron microscopy were performed after explantation. RESULTS: Fifty percent of the grafts occluded within two weeks. One animal died with patent grafts after 14 days. In the three remaining animals 5/6 grafts were patent after nine months. Two animals were followed 13 months after implantation with 3/4 grafts patent at explantation. All patent grafts had confluent endothelial-like cells. CONCLUSIONS: Biosynthetic small calibre vascular grafts made from BC can be patent for up to 13 months in sheep carotid arteries. BC is a potential material for small calibre grafts but patency in animal models needs to be improved before clinical studies can be planned.

Nanocellulose (bacterial cellulose, BC), such as that produced by Acetobacter xylinum, has shown promising results as a replacement material for small diameter vascular grafts. The surface morphology of the lumen and mechanical properties of such tubes are crucial for their performance. The growth of a BC tube in a vertical fermentation bioreactor using silicone tubing for support and as an oxygen delivery membrane has not been studied in detail previously. Oxygen concentration and the number of bacteria added influence the production of the BC tubes. A dense and smooth luminal surface was formed after 4 days on a 3 mm silicone support. The bacteria were found to be in high concentration close to the silicon support and decreased in number further away. In the region with a high bacteria concentration, dense thin layers of BC were formed since the bacteria moved close together in this region. The presented observations were summarized in a theoretical model of BC tube growth.