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CHECK OUT THESE NATURE OUTLINES,SUPPORTED BY TRITRI has partnered with Nature Outline to bring
you a series of informative collections covering
the latest research in the medical sciences.

CRITICAL LIMB ISCHAEMIA OUTLINEOUTLINE CRITICAL LIMB ISCHAEMIASAVING LIFE AND LIMB

WATCH AN ANIMATION AT:GO.NATURE.COM/2WDOZTLA PROGRESSIVE DISEASE

Up to 30% of people4 with CLI are unable to undergo
surgical treatments for the condition because their arteries
are too calcified or they have another condition that prevents it.
As an alternative, researchers are investigating whether new
blood vessels can be encouraged to grow, thereby restoring thesupply of blood to the affected limb. Gene and cell-based therapies
are the main regenerative-medicine approaches being pursued.

More than

50%

Critical limb ischaemia (CLI) is the most severe form of a chronic
cardiovascular condition called peripheral arterial disease, which isusually caused by atherosclerosis. Although CLI notably affects thelimbs, the majority of patients will die from a heart attack or stroke1.

1

GENE THERAPYGROWING PAIN

2

34

56

Relative risk of CLI

CURRENT TREATMENTS
3

The gold standard in treatment for
CLI is surgery to unblock or widen
the obstructed blood vessel, or tobypass it through another vessel.

CLI is usually the end stage of
peripheral arterial disease, in which
a limb is starved of blood. The
onset can be sudden if aplaque in a major artery
ruptures, causing a bloodclot that rapidly andseverely restricts
blood flow.

VEGF

2Vectorenters cell
Viral vector

4

Hypertension
1Nucleus

VEGF protein
promotesgrowthof newbloodvessels

Cells from the patient are used to supply substances that promote
blood-vessel formation and boost the growth of new arteries to theaffected limb. Cell therapy seems to provide a more sustained andtargeted release of such substances than gene therapy.

Diabetes

Narrowed
arterySmall phase I and phase II clinical trials have shownthat cell-based therapies are safe and improve woundhealing, but the trials were not large enough to detect any
improvements in delaying amputation4.

1

2

Blood is collected fromthe patient and separated
into its component parts
using a centrifuge.

Mononuclear cells are
injected into the affectedlimb. These target sites oftissue damage, regenerating
the lining of blood vessels andhelping to form new vessels.

Blood-vessel growthallows blood to bypass
the blockage.

Blood vessels
grow into theaffected limb,
improving theblood supply.

PlasmaEndovasculartreatment toremove plaqueor widenvesselRupture
Blood clot

VEGF gene
inserted intocell’s DNA

Endothelialcell

CELL THERAPY

SmokingOver time, the plaque hardens andnarrows the lumen of the artery.
The patient experiences agradual progression ofsymptoms, including
tingling, numbness andpain in their limbs.

1
VEGF gene
inserted intovirus genome

So far, gene therapies have been shown
to improve wound healing in patients, but
do not considerably prolong the length of time between
diagnosis with CLI and limb amputation5. Targeting thecorrect cells is crucial because promoting blood-vessel
formation in healthy tissues might increase the riskof tumours.

In Europe and the United States,
there are 500–1,000 new cases of
CLI per 1 million people each year3.
The risk of developing CLI isincreased for patients with certain
risk factors, including hypertension,
diabetes and smoking1.

Bloodflow3

A modified virus or other vector is used
to insert genes into the cells that line theinner wall of the blood vessel. These genes
encode proteins, such as vascular endothelial
growth factor (VEGF), that promote the growthof new blood vessels.

mortality within 5 yearsof CLI diagnosis2.

Arterywall

In atherosclerosis, the body's
arteries are affected by abuild-up of plaque madeof substances found in
the blood such as fat,cholesterol and calcium.
Plaque2

SAVING LIFE AND LIMBRESCUE THROUGH REGENERATION

Many people with critical limb ischaemia have no option but to havethe affected limb amputated. Can regenerative medicine offer an alternative?
By David Holmes; illustration by Lucy Reading-Ikkanda

Mononuclear cells
Injection
sites

Centrifugation
mediumWhite blood cells
and platelets

AffectedfootEPC

Red blood cells

EXTREME SYMPTOMSCD34NEXT STEPSThe effects of CLI areusually most severe in
the extremities of theaffected limb.

Efforts to optimize cell-based therapies for CLI are under way. One approach is to increase the proportion of a typeof mononuclear cell called endothelial progenitor cells (EPCs), which have a role in blood-vessel regeneration, in theinjected cells. This is achieved by selecting cells that express the protein CD34 on their surface. A small 2009 trial6
showed that people who received such CD34-expressing cells showed improvements in pain scores and in thedistance that they could walk. A larger trial is being planned in Japan for later this year to test whether the therapy
can delay or prevent the need for amputation.

Gangrene
Non-healing ulcer

Arterybypass

http://www.nature.com/nature/outline/critical-limb-ischaemia

Sources: 1. Davies, M. G. Methodist Debakey Cardiovasc. J. 8, 10–14 (2012). 2. Teraa, M., Conte, M. S., Moll, F. L. & Verhaar, M. C. J. Am. Heart Assoc. 5, e002938(2016). 3. Norgren, L. et al. J. Vasc. Surg. 45, S5–S67 (2007). 4. Samura, M. et al. J. Transl. Med. 15, 49 (2017). 5. Belch, J. B. et al. Lancet 377, 1929–1937 (2011).
6. Kawamoto, A. et al. Stem Cells 27, 2857–2864 (2009).

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NON-UNION BONE FRACTURE OUTLINEOUTLINE NON-UNION BONE FRACTURE

CLOSING THE GAPWATCH AN ANIMATION AT:
GO.NATURE.COM/2XZNFE1

Around 5–10% of bone
fractures fail to heal1.
As the populations ofdeveloped countries age,the incidence of fractures,
including non-union bone
fractures, will increase.

Brokenfemur

STATE OF THE UNION

In the United Kingdom,
the incidence of fractures2 ishighest in men aged 15–24
and in women older than 85.

INTO THE BREACH

SOFTLY DOES IT

TOUGHENING UP

0–2 WEEKS

2–6 WEEKS

6–12 WEEKS

Immediately after fracture,
blood from ruptured vessels
inside the bone starts tocoagulate into a swollen masscalled a haematoma. At thisearly stage, it is crucial that thefragments of bone are guided
back into place to ensure thatthey are correctly aligned and tohelp stem the loss of blood.

A soft callus starts to form,
made up of new connective
tissue, microscopic bloodvessels, cartilage and soft
spongy bone.

By 12 weeks after fracture, osteoblasts havetransformed the soft callus into a hard callus.
The length of the healing period depends onthe fracture site — bones in the lower limbs
can take longer because they are larger.

Magnetic
particle

Chondrocyte
Osteoblast

2
CD34

MagnetBlood vessel

43Magnetic
filtration
purifies themixture ofcells.

Mononuclear cells
expressing CD34 aretagged with magnetic
particles.

1

Mononuclear cells
are extracted fromthe patient’s blood.

EPCs

The healed bone will continue to be
remodelled over the coming months
and years, in response to stresses imposedby physical activity.

FibroblastAt least

400,000of thesefractures will
fail to heal.

On days 1–5 of treatment,
the patient is injected witha cytokine that prompts
mononuclear cells tomigrate from the bonemarrow into the blood.

They aim to deliver a type of mononuclear cell called endothelial progenitor
cells (EPCs) to the fracture site to induce blood-vessel regeneration andpromote bone formation. EPCs express the protein CD34 on their surface, whichacts as a marker that enables them to be identified in and extracted from blood.

SNAP TO IT

Almostfractures arereported each year in the United States3.

The age beyond
which women’s riskof fracture increases
steadily2 — probably
owing to a loss ofbone density.

Conventionally, the treatment of non-union bone fracture involves surgery tostabilize the fracture site and bone grafting to stimulate the growth of bone andnew blood vessels. Researchers are now turning to regenerative medicine tohelp heal such fractures.

Although most fractures will heal fully, a small but notable proportion do not. These severe cases— known as non-union bone fractures — can cause long-term disability and pain, and often
require surgery to rectify. To determine why non-union bone fracture occurs, it is important tounderstand how bone healing typically progresses.

8 MILLION

55

BREAKING POINT

A bone fracture that fails to heal after initial treatment can lead toprolonged disability. Regenerative therapies might help to restart thebone-healing process, getting the people affected back in action.
By David Holmes; illustration by Lucy Reading-Ikkanda

Cells build
scaffold
between bonefragments

Critical limb ischemia is a
painful arterial disease that
restricts blood flow and canresult in the loss of a limb.
To find out more read Nature
Outline: Critical limb ischemia:

Bonemarrow

Mononuclear
cellFiltrationand sorting

New bloodvessel growththrough softcallus

Bone

Connective
tissue andcartilage

Haematoma

Hardcallus
Graft fromhealthybone

Soft callus

Yellow bonemarrow

Gelscaffold
Expansion ofred bonemarrow
towards areaof damage

Red bonemarrow(white blood celland red blood
cell production)

567

Cells expressing CD34 areinfused into a gel scaffold.

The cell-filled scaffold is
implanted at the fracture
site, together with apiece of healthy bone.

The implanted cells promote blood-vessel growth.
These CD34-expressing cells can also transform intoother types that can form blood vessels or bone.

UNHAPPY MARRIAGE
6–9 MONTHS
Non-union bone fracture is diagnosed if there isno evidence that the broken bone will heal fully
without further medical intervention.

NEXT STEPSLimited swelling
Soft connective tissue fails
to mature into bone
Limited blood supply

Diabetes

Anti-inflammatory, opioidand anticoagulant drugs

RISK FACTORS

Arthritis

Healedfemur

Some fractures don’t respond tostandard treatments, leading tolasting pain and disability. To findout more read Nature Outline:
Non-union bone fracture:https://www.nature.com/nature/journal/v550/
n7677_supp_out/full/550S194a.html

Sources: 1. Mills, L. A. & Simpson, A. H. R. W. BMJ Open 3, e002276 (2013). 2. Donaldson, L. J., Reckless, I. P., Scholes, S., Mindell, J. S.
& Shelton, N. J. J. Epidemiol. Community Health 62, 174–180 (2008). 3. Buza, J. A. III & Einhorn, T. Clin. Cases Miner. Bone Metab. 13, 101–105(2016). 4. Kuroda, R. et al. Stem Cells Transl. Med. 3, 128–134 (2014).

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S 1 9 4 | NAT U R E | VO L 5 5 0 | 2 6 O C T O B E R 2 0 1 7Severe or complex
fracture

A 2014 study4 by researchers in Japan showed that treatment with CD34-expressing cells seems to be safe,and bone fractures healed in all people who received the therapy. But the study was too small to determine any
clinical benefit. A larger, three-year trial at several institutions to study the effects of CD34-based therapy startedin 2015. The results should reveal whether regenerative medicine can help to heal broken bones.

CLOSING THE GAP

The TRANSLATIONAL RESEARCH INFORMATICS CENTER, or ‘TRI’, provides comprehensive support
programmes for medical researchers for all phases of clinical study, from research planning to data analysis.
By supporting Japan’s best and brightest to create breakthrough innovations in technology and medicalscience, TRI is working towards tangibly improving the prognoses of intractable human diseases.
SUBTLE PATTERNS © TOPTAL DESIGNERS