Ny forskningsrapport från England

Idag 21/11 publicerar den vetenskapliga skriften Hindawi Stem Cells International en studie utförd av 6 brittiska forskare. Received 6 September 2019; Accepted 1 November 2019; Published 21 November 2019

Studien handlar om stamcellsforskning inom området frakturläkning. Ur abstractet får vi vägledning.

Colony Formation, Migratory, and Differentiation Characteristics of Multipotential Stromal Cells (MSCs) from “Clinically Accessible” Human Periosteum Compared to Donor-Matched Bone Marrow MSCs

Abstract
Periosteum is vital for fracture healing, as a highly vascular and multipotential stromal cell- (MSC-) rich tissue. During surgical bone reconstruction, small fragments of periosteum can be “clinically accessible,” yet periosteum is currently not ultilised, unlike autologous bone marrow (BM) aspirate. This study is aimed at comparing human periosteum and donor-matched iliac crest BM MSC content and characterising MSCs in terms of colony formation, growth kinetics, phenotype, cell migration patterns, and trilineage differentiation capacity.
“Clinically accessible” periosteum had an intact outer fibrous layer, containing CD271+ candidate MSCs located perivasculary; the inner cambium was rarely present.
Following enzymatic release of cells, periosteum formed significantly smaller fibroblastic colonies compared to BM (6.1 mm2 vs. 15.5 mm2, , ).
Periosteal colonies were more homogenous in size (range 2-30 mm2 vs. 2-54 mm2) and on average 2500-fold more frequent (2.0% vs. 0.0008%, , ) relative to total viable cells.
When expanded in vitro, similar growth rates up to passage 0 (P0) were seen (1.8 population doublings (PDs) per day (periosteum), 1.6 PDs per day (BM)); however, subsequently BM MSCs proliferated significantly slower by P4 (4.3 PDs per day (periosteum) vs. 9.3 PDs per day (BM), , ). In early culture, periosteum cells were less migratory at slower speeds than BM cells.
Both MSC types exhibited MSC phenotype and trilineage differentiation capacity; however, periosteum MSCs showed significantly lower (2.7-fold) adipogenic potential based on Nile red : DAPI ratios with reduced expression of adipogenesis-related transcripts PPAR-γ.
Altogether, these data revealed that “clinically accessible” periosteal samples represent a consistently rich source of highly proliferative MSCs compared to donor-matched BM, which importantly show similar osteochondral capacity and lower adipogenic potential.
Live cell tracking allowed determination of unique morphological and migration characteristics of periosteal MSCs that can be used for the development of novel bone graft substitutes to be preferentially repopulated by these cells.

Studien berör de patienter med benbrott som inte läker ihop på naturlig väg. Upp till 10% av alla benbrott kan kroppen själv inte reparera och de patienter det berör behöver då undergå ett kirurgiskt ingrepp där man exempelvis sätter in en metallplatta,skruvar,div andra mojänger för att fästa ihop delar av kroppens egna ben.
Ett vanligt ingrepp är där man "spikar" ihop en höftled.Utan ett operativt ingrepp får patienten en betydande livskvalitetsförsämring vilket är lätt att förstå. Ingrepp av dessa typer är kostsamma sett till material som kvalificerad personal för att klara själva operationen. 80% av de som opererats med denna metod anses lyckosamma då de får ett hyfsat bra liv efter ingreppet. För de 20% som kräver omoperation är prognosen inte lika bra.
Men tillbaka till aktuell studie.
Dessa 6 forskare har studerat anledning till att ben inte läker ihop naturligt utan kräver ingrepp.
Man vet sen tidigare att benbrottspatienter som dessutom är diabetiker eller rökare är överrepresenterade bland de som inte läker ihop.
Utifrån detta kan man som lekman (jag) lista ut att blodförförsörjning är gemensamt för bägge grupperna.
Lägre syresättning av blodet alltså.
Även forskarna har utgått från detta och fokuserat på vad det innebär för själva läkningsprocessen.
Man vet sen tidigare att de hinnor som omsluter ben är viktiga för läkning.
Benhinnor, Periosteum på latin, är definierat som ett tunt yttre membran som omger ett ben.
Det innehåller bindväv, kapillärer, nerver och ett antal olika celltyper.
Från studien : "The critically important role of periosteum during bone fracture healing is well established."

Forts...
Forskarna har med stamcellsforskning försökt komma förbi det problematiska att en benhinna från ena benet inte "kopplar" samman med hinnan till det ben som ska läkas samman med.
Och man har med denna studie hittat tekniken som man tror kommer "koppla" ihop benhinnorna.
De har använt sig av LIVE CELL TRACKING (Imaging) för att följa en specifik komponent i en stamcell på en frisk benhinna.Närmare bestämt "the cambium layer",på svenska kambiumskiktet.

"Periosteum lines the outer aspect of most bones, contributing to appositional growth during childhood and adolescence as well as providing the vascular supply to bone [14]. This tissue, although thin, forms two distinct layers, the bone lining inner cambium layer being highly cellular and containing osteogenic progenitors, while the outer fibrous layer, predominantly consisting of collagen, is highly vascular but not particularly cellular in comparison [12, 17]. The cambium layer is tightly attached to the underlying bone by Sharpey’s Fibres; thus, removal of periosteum with the cambium layer attached is difficult [18] and is furthermore considered inappropriate by the surgeons as potentially compromising the repair process. However, “clinically accessible” samples of periosteum, likely to be fibrous in nature, can be obtained as by-products of surgical debridement of the fracture site, but their osteogenic progenitor cell content has not yet been explored before."

Kambiumskiktet vet man sen tidigare har stor betydelse vid läkningsprocessen.
Detta skikt (celler) har man isolerat  från en donators friska stamcell och jämfört med samma skikt (celler) hos en benbrottspatient.Sen har man följt bägges celler över tid genom ett HoloMonitor mikroskop och sett hur benbrottspatientens celler gradvis tappat/minskat sina läkande egenskaper.
Det forskarna kommit fram till är att om man förstärker läkningen med donatorns tillförda celler från kambiumskiktet ökar läkningsprocessens möjlighet till full läkning.
Man skriver att med transplantation av dessa celler till ett komplicerat benbrott är det ett alternativ till dagens använda lösningar.
 - "This would allow for periosteum as a proliferative “MSC-rich” source to be transplanted into or around defect areas for the treatment of complex fracture or critical size bone defects, as a conjunct with other healing stimulating factors and cell sources that are currently in use."

Jag återkommer här till HUR de bar sig åt,med vilken teknik de kom fram till denna lösning.
Ni vet förmodligen vart jag är på väg. 😎
Så här kommer urklippta partier från studien.Fetningarna är mina.

Materials and Methods

2.6. Live Holographic Imaging

P0 vials of donor-matched (male, 17) BM and periosteum MSCs were seeded in duplicate (250 cells per well) onto a Lumox® multiwell 24-well cell culture plate (Sarstedt). The cultures were grown for 3 days at 37°C, 5% CO2 in StemMACS media. On day 3, following a half medium change, the plate lid was replaced with PHI HoloLids™ imaging covers (PHI, phase holographic imaging) (sterilised in 70% EtOH, 10 mins). The plate was placed onto the xy motorised stage of a HoloMonitor M4 Microscope, set up inside an incubator. Using the Hstudio software, three coordinates in each well were manually focused and set to automatically image every hour for 2 days.

Post acquisition, the Hstudio software applied a “mask” to the images, where a threshold was set to distinguish cells from the bottom of the well, allowing for automatic cell identification. Automatic cell number assignment allowed for individual cell tracking over time, which was checked manually. All outputs were exported into Excel files and plotted using Prism.

2.7. Statistical Analysis

To compare donor-matched periosteum and BM samples, statistical analysis was carried out using appropriate paired tests (Wilcoxon signed-rank test or paired test depending on data distributions), where P < 0,05 was considered significant.
Phase holographic imaging data sets were compared using an unpaired Student’s -test.
The exact tests are specified in the figure legends, and the data is presented mean +/- standard deviation.

3.3. Live Cell Tracking of Periosteum and Bone Marrow MSC Cultures

To explore if the observed differences seen in colony size between periosteum and BM MSCs could be due to different cell migration patterns, live cell imaging and tracking of cell movement over time was carried out using holographic imaging.

The cell migration and movement patterns of periosteal and BM MSCs were assessed by live tracking of individual cells.

4. Discussion

In this study, the potential for the utilisation of periosteum-derived MSCs as a source of MSCs during bone defect surgical repair was investigated. Currently, there is a lack of direct comparisons of MSCs derived from human periosteum and BMA, especially when donor matched [24, 35]. Here, for the first time, donor-matched comparisons of “clinically accessible” periosteum (from within the surgical opening of a fracture site) were compared to iliac crest BMA, a supply of MSCs currently used in surgery [36].

Both MSC sources were compared histologically to locate CD271+ candidate MSCs within each tissue [21, 37–39]. The cambium layer of periosteum has been historically described as a rich source of osteoprogenitors, whereas the fibrous layer is thought to be less cellular [16, 17, 40, 41]. However, this study has shown the presence of CD271+ cells in human samples located throughout the outer fibrous layer. These cells were localised surrounding blood vessels throughout the fibrous layer; therefore, even though the cambium layer was often not harvested, these data indicated that “clinically accessible” human periosteum could still contain a supply of MSCs.

In order to underpin the differences seen in colony formation between periosteum and BM MSC cultures, live cell imaging of early in vitro (2 weeks in culture) MSCs was carried out.
Phase holographic imaging was utilised as a noninvasive, unlabeled high throughput method for quantifying cellular morphology and live cell tracking [46–48]. MSC morphology in both MSC sources was variable over time, changing hour by hour, with approximately 98% of the cells measured retaining the classical “spindle” shape, and the remaining 2% of the measured cells were retracted into spheres, undergoing division.

5. Conclusions

“Clinically accessible” samples of long bone periosteum, a vital component of bone fracture repair has been shown to be a rich source of highly proliferative MSCs, compared to the current “gold-standard” BMA, with lower adipogenic but similar osteochondral potential.
A novel live cell tracking technique permitted extensive quantification of morphological and migratory characteristics of periosteal MSCs that can be used to inform the development of novel bone graft substitutes to be repopulated by these cells. Further investigation into “minimally manipulated” periosteum samples, for example, periosteal micrografts, is needed for future clinical translation of this tissue source for use during single surgical procedure.

Min kommentar
Det forskarna skriver i slutordet "A novel live cell tracking technique" är alltså att denna nya teknik (HoloMonitor) möjligjorde den omfattande kvantifiering av morfologiska och migrerande egenskaper denna process (periosteal) krävde för att komma till dessa nya insikter.
Bra så,eller hur? Ett nytt forskningsområde där HoloMonitor visar sina unika egenskaper !
I en förlängning kanske denna studie, där HoloMonitor varit så central, kommer leda till att ersätta alla operationer  med metallbitar,titanskruvar och andra mojänger för benbrottspatienter? Nåt pris för den bedriften borde i så fall PHI/Holomonitor få.Kanske inte i Nobelklass,men snäppet under.

De 6 engelska forskarna har bedrivit sina studier vid Leeds University.Närmare bestämt fakulteten

Leeds Institute of Rheumatic and Musculoskeletal Medicine

Edit. 22/11
En uppmärksam PHIare kommenterade att Leeds Uni troligtvis är en befintlig kund och alltså inte en ny dito.
Så bloggen får plocka bort grädden på detta mos.Men istället avslöja att vi snart kommer få ta del av en annan stamcellsforskningsrapport.Även den handlar om ben,närmare bestämt benvävnad.
Det är kinesiska forskare som ligger bakom studien och den är tyvärr låst för tillfället,men snart lär man öppna upp den aningens mer för att komma åt Holo-infon.När det sker kommer bloggen naturligtvis publicera det material som forskarna behagar släppa.

KLF2⁺ stemness maintains human mesenchymal stem cells in bone regeneration

                          Mvh en nattjobbande the99

Ps. lite "uppskattning" via tröckeri på prytteln nedanför kanske grävjobbet är värt?