Bone is often considered as a rigid organthat provides support and physicalprotection to various vital organs of the

body. In reality, bone is a very dynamic organ thatis permanently in a dynamic balance, a processcalled ‘remodelling’, which allows a constantregeneration of bones. In the adult human body thistightly regulated remodelling process allows for therenewal of the entire skeleton every seven years.

Additionally, bone has also other importantproperties: it stores crucial nutrients, minerals,and lipids, and it produces blood cells thattransport oxygen and play a vital role in protectingthe body against infection.

Bone remodelling is only possible through acomplex co-ordination of multiple bone marrowcell types: bone formation by osteoblasts andresorption by osteoclasts. An imbalance betweenbone formation and resorption can result invarious diseases, such as osteopetrosis,osteopenia, and osteoporosis.

Bone fracture healingOne of the most fascinating properties of bone isthat unlike in other tissues, the majority of bonyinjuries (fractures) heal without the formation ofscar tissue, and bone is regenerated with its pre-existing properties largely restored and withthe newly-formed bone being indistinguishablefrom the adjacent uninjured bone.

The process of fracture healing byintramembranous ossification and/or endochondralossification also involves many events including thesignalling, recruitment, and differentiation of bonemarrow stromal cells during the early phase; theformation of a hard callus and extracellular matrix,angiogenesis and revascularisation during the mid-phase; and finally callus remodelling at the latephase of fracture healing.

Impaired healingDespite the fine degree of orchestration duringfracture healing, the process may be impaired.Currently, 10–15% of the fractures that occur

annually result in poor or unresolved healing, so-called ‘non-unions’. The mechanism ofimpaired osteoporotic fracture healing is multi-factorial and depends on the low sensitivityof osteoblasts to mechanical signals, reducedangiogenesis, and decreased amounts ofmesenchymal stem cells. Particularly problematicand a major clinical orthopaedic challenge arethe so-called ‘critical size defects’, which arebone fractures producing a gap that is not ableto completely heal alone over a long period. Inthese cases, it is necessary to fill the non-uniondefect with dedicated materials or alternativelyuse strategies, which promote completeregeneration of the bone in these defects.

Nevertheless, current clinical treatments can beproblematic and often yield poor healing due tothe anatomy and physiology of bone tissue, aswell as due to the limited knowledge about thebone healing process itself.

Bone: a dynamic organ

Professor Paolo Cinelli discusses the identification of stem cells forthe development of bone bioengineering technologies

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The gold standardThe gold standard is the transplantation ofcancellous autogenous bone, a procedure thathas some drawbacks for clinical applications,such as limited availability, morbidity, and donorsite pain.

Skeletal defects may require volumes of bone thatare often not available. Therefore, allografts arealso used as substitutes for autologous bonegrafts, but often are not sufficient to solve themany problems of bone deficiency. Alternatively,biocompatible materials can be used (e.g. calciumphosphate bioceramics) but, unfortunately, to dateno single synthetic material offers all the benefitsof the patient’s own bone.

These materials can also be used in combinationwith osteoinductive factors like bonemorphogenetic proteins (BMPs), transforminggrowth factor-α (TGF-α), and fibroblast growthfactor (FGF). Due to the high number of limitationslinked to the above-described procedures and thesteadily increasing demand for bone graftingprocedures, it is essential to develop bettertherapeutic approaches.

Bone tissue engineeringIn this context, tissue engineering combining theuse of stem cells with synthetic scaffolds andmolecular signals (growth or differentiatingfactors) to form hybrid constructs represents afascinating alternative.

Stem cells represent an appealing cell source forregenerative medicine applications aimed atimproving bone fracture healing and treatingbone-remodelling diseases like osteoporosis.Many reports have described the presence ofmesenchymal stem cells in a variety of foetal,perinatal and adult tissues, including peripheralblood, foetal liver and lungs, skeletal muscles,amniotic fluid, synovium and the circulatorysystem where they contribute in maintenance oftissue homeostasis. These cells are able todifferentiate in vivo as well as in vitro, under theright culture conditions, into osteoblasts,chondrocytes, and adipocytes.

In parallel to mesenchymal stem cells, a numberof additional stem cell populations with pluripotentand multipotent properties exist which canpotentially be used for bone engineering, such asembryonic stem cells, induced pluripotent stemcells, and adult neural crest stem cells.

Mesenchymal stem cellsIn recent years, human fat tissue has beenidentified as a good source of mesenchymal stemcells, the so-called ‘adipose-derived stem cells’.These are isolated from the stromal vascularfraction of adipose tissue, which contains pre-adipocytes, mesenchymal stem cells,endothelial progenitor cell, T- and B-cells, mastcells as well as adipose tissue macrophages. Theamount of stem and progenitor cells found in the

uncultured SVF from adipose tissue was estimatedto be around 3% of the whole cells. Thiscorresponds to 2,500-fold more stem cells thanin the bone marrow. These cells representtherefore a good alternative to BMSCs for boneregeneration purpose.

The main problem with mesenchymal stem cellsof both bone marrow and fat tissue is that theyconsist of highly heterogeneous populations ofstem and progenitor cells. Therefore, describingthe entire culture as stem cells is inappropriate.

Moreover, several studies have reported differencesin the expression of surface markers onmesenchymal stem cells and the currently-usedmarkers are not a unique characteristic ofmesenchymal stem cells; rather they reflect theirheterogeneity. This variability represents a limitingfactor for the efficient use of mesenchymal stemcells in the clinic. Thus, for the clinical use ofmesenchymal stem cells for regenerationpurposes, a better characterisation of the cells anda standardisation of the isolation and cultureprotocols are urgently required. To this end, it ismandatory to dissect at clonal level the compositionof the different populations which are obtained withthe current protocols.

Molecular mechanismsOur laboratory has, for several years beeninterested in dissecting the molecular mechanismsinvolved in the maintenance of stem cell identityin different stem cell populations, like embryonicstem cells, induced pluripotent stem cells andmesenchymal stem cells. The goals of our projectsare, in addition to the study of the molecular

mechanisms underlying stem cell identity, toidentify populations of multipotent stem cells whichcan be prospectively used for the development ofbone bioengineering technologies.

Higher osteogenic potentialThe isolation and analysis of mesenchymal stemcells subpopulations with higher osteogenicpotential is also an important area.

The use of highly selective cell separationprocedures in clinical cell-based treatments hasthe potential to improve the quality of repair andthe subsequent clinical outcome. Nevertheless,the methodologies have to be adapted to aclinically compatible asset.

The cultivation of cells for therapeutic applicationsimplicates the use of good clinical practice (GCP)facilities and long-time expansion of the cells invitro before transplantation. It would therefore beinteresting to develop a protocol in whichsubpopulations of stromal vascular fraction cellswith a higher osteogenic differentiation potentialcould be enriched and employed without furtherexpansion in vitro for clinical applications.

Magnetic activated cell sortingAn optimal solution would be the possibility ofperforming the enrichment directly in theoperation room in parallel to the alreadynecessary operative treatment of the patients (seeFig. 1). An interesting technology for this purposeis magnetic activated cell sorting (MACS).

In a recent study, we aimed at testing thefeasibility of such an approach by using pericytes.Pericytes or vascular stem/precursor cells at

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Fig. 2 In the adult human body the tightly regulated remodelling process, which allows a constantregeneration of bones, allows for the renewal of the entire skeleton every seven years

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different stages of differentiation are located inthe wall surrounding the vasculature. Previousstudies have identified markers typifying pericytes,in particular the surface markers NG2 and CD146(also known as MCAM or S-endo1), which can beused to isolate pericytes upon depletion of theCD34 positive endothelial cell population and ofthe CD45 positive haematopoietic cells.

To this end, we have enrichedCD146+NG2+CD45– pericytes from the stromalvascular fraction of fat tissue by magnetic sortingand tested their capacity, without any in vitroexpansion, to take part in bone regeneration. We were able to show that enrichedCD146+NG2+CD45– cells are able to differentiateto osteoblasts and induce calcium deposition(mineralisation) with a higher efficiency asunsorted adipose stem cells.

We further tested the regenerative capacity of thecells in a mouse model for femoral segmentalcritical-sized defect. In this model, a 3.5-mm-longsegmental bone defect is induced in the mid-shaftof the mouse femur and bone fixation is performedwith a titanium microlocking plate with four lockingscrews. The freshly isolated cells were seeded ona collagenous bone scaffold and the scaffold wasinserted into the segmental bone gap.

Eight weeks after the operation the bones wereisolated and following the removal of the platesthe bone were analysed by microcomputertomography and histologically. From this, wecould confirm that direct transplantation ofCD146+NG2+CD45– enriched cells fromadipose tissue is sufficient to promote in vivobone regeneration.

This study represents proof of principle for the useof enriched populations of cells withstem/progenitor identity for direct therapeuticapplications and opens a new perspective forusing stem cells in a clinical setting.

Stem cell maintenanceThe identification of factors essential for themaintenance of stem cells. Pluripotent stem cellsharbour a powerful potential for therapeuticapplication because they are able to generateevery cell type of the body. Although it is possibleto reprogramme somatic cells to a pluripotentstate, the mechanisms underlying maintenanceand determination of pluripotency remain unclear.

The same holds true for the mechanism drivingthe differentiation of these cells. This is not onlythe case for pluripotent stem cells but also ingeneral for a large number of adult stem cells.Even though their identity is known, it often isimpossible or difficult to maintain them outside ofthe body in a self-renewing pluripotent state.

Our laboratory has invested many efforts into theidentification of new factors important for themaintenance of pluripotency and in analysing themechanisms driving the reprogramming ofsomatic cells. We have, for example, elucidatedthe role of role of poly-ADP-ribose polymerase 1(Parp1) during reprogramming and found thatpoly-ADP-ribosylation of the reprogrammingfactor Sox2 by Parp1 plays an important roleduring the first days upon transduction with theYamanaka reprogramming factors.

We were also able identify Pramel7 (preferentiallyexpressed antigen in melanoma like-7) as a novel

factor crucial for LIF-mediated self-renewal inembryonic stem cells and recently show thatPramel7 targets UHRF1, a key factor for DNAmethylation maintenance, for proteasomaldegradation. This process leads to a genesignature and DNA hypomethylation comparableto the preimplantation epiblast, the developmentalground state and source of embryonic stem cells.

Our discovery revealed a previously unknowndynamic nature of DNA methylation throughproteasome pathways and helps to improveembryonic stem cell culture to reproduce in vitrothen in vivo ground-state pluripotency.

What is the true identity ofmesenchymal stem cells?A vast number of studies have identified a varietyof cell surface markers expressed bymesenchymal stem cells in hopes of developingmethods to isolate them more efficiently. However,these markers are not specific, either individuallyor in combination, and importantly theirexpression changes with time in culture. The realidentity of mesenchymal stem cells is thereforelargely unknown.

Nevertheless, this knowledge is of utmostimportance to answer such basic questions aswhether the trilineage potential of mesenchymalstem cells (osteogenic, chondrogenic andadipogenic differentiation) is an intrinsic propertyof a specific subtype of stem/progenitor cells ordifferent types of stem/progenitors cellsdifferentiate toward different fates. Thisinformation is not only essential for bonebioengineering but also for understanding the roleof stem cells in diseases like osteoporosis.

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Fig. 3 Stem cells represent an appealing cell source for regenerative medicine applications aimed at improving bone fracture healing and treatingbone-remodelling diseases like osteoporosis

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We have recently developed a new tool based onthe novel technology of cytometry by time-of-flight(CyTOF) to dissect the heterogeneity of humanmesenchymal stem cells. With this tool, we are able to perform real-time analysis at high-dimensional level and at single cell resolutionto detect the distribution and changes of markerswithin the heterogeneous population ofmesenchymal stem cells.

In combination with state-of-the-art omics andimaging technologies we aim at dissecting thecomposition at single cell level of stromal vascularfraction cell populations and identify new specificmarkers for the prospective identification andisolation of selected stem cell populations forbone bioengineering.

BibliographyKonig, MA, Canepa, DD, Cadosch, D, Casanova, E,Heinzelmann, M, Rittirsch, D, Plecko, M, Hemmi, S, Simmen,HP, Wanner GA, Cinelli, P (2016). Direct transplantation ofnative pericytes from adipose tissue: A new perspective tostimulate healing in critical size bone defects. Cytotherapy 18,41-52.

Graf, U, Casanova, EA, Wyck, S, Dalcher, D, Gatti, M,Vollenweider, E, Okoniewski, MJ, Weber, FA, Patel, SS, Schmid,MW, et al. (2017). Pramel7 mediates ground-statepluripotency through proteasomal-epigenetic combinedpathways. Nature Cell Biology 19, 763-773

Weber, FA, Bartolomei, G, Hottiger, MO, and Cinelli, P (2013).Artd1/Parp1 regulates reprogramming by transcriptionalregulation of Fgf4 via Sox2 ADP-ribosylation. Stem Cells 31,2364-2373

Casanova, EA, Shakhova, O, Patel, SS, Asner, IN, Pelczar, P,Weber, FA, Graf, U, Sommer, L, Burki, K, and Cinelli, P (2011).Pramel7 mediates LIF/STAT3-dependent self-renewal inembryonic stem cells. Stem Cells 29, 474-485

Professor Paolo CinelliCABMMUniversity of Zurichhttp://www.cabmm.uzh.ch/en/Membership2/MemberApplFields/MolMed/PaoloCinelli.html

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Fig. 4 Uncovering the real identity of mesenchymal stem cells is not only essential for bonebioengineering but also for understanding the role of stem cells in diseases like osteoporosis

Smart implants Innovative and personalised orthopaedic implantsare complex, requiring cross-disciplinaryknowledge and understanding of processes suchas 3D printing technology, computer modelling,and regenerative medicine, amongst other areas.Moreover, the current generation of implants havea limited lifespan – around 15 years – meaningthat they need to be replaced up to twice in apatient’s life through the means of revisionsurgery. As revision surgery is a complicatedprocedure, it can often lead to bone defects,whereby standard treatment to repair thesebones involves using the patient’s bone, orsynthetic filler materials, in combination withmedical implants. These complex proceduresare long and can result in a high risk of infection,whether immediately after surgery or furtherdown the line. Treatment for infection is also achallenging and extended process, which isoften associated with hospitalisation and theincurrence of high healthcare costs.

In order to address problems with currentimplants, calls are being made for next-generation smart medical implantsintended to accelerate healing whilstpreventing infection. It is here that PRosPERoS(PRinting PERsonalized orthopaedic implantS)comes in, a project funded through InterregVA Flanders, the Ministry of Economic Affairs,and Provinces Limburg (the Netherlands), as well as Flemish-Brabant (Belgium).PRosPERoS intends to develop personalised,smart, and biodegradable implants which arecreated through 3D printing on magnesiumand zinc alloys. The process involvesconducting a precision scan of the vertebraewith advanced imaging technology, as a resultdesigning and printing implants in a waywhich is patient-centric.

Meanwhile, in an effort to reduce the high ratesof osteoporosis in North Western Europe –leading to the greatest rates of fracturesthroughout all European regions – the BONE(Bio-fabrication of Orthopaedics in a New Era)project is looking to regenerative medicine tocreate smart implants. BONE aims to acceleratethe uptake of cost-effective 3D smart implants,which are fabricated through electrospinning(ESP) technology. The technology supportsregeneration of skeletal bone whilst replacingthe demand for tissue donors, revision surgery,or lifelong medication schedules.

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