Mesenchymal Stem Cells for the Treatment of Spinal Arthrodesis:...

Review ArticleMesenchymal Stem Cells for the Treatment of SpinalArthrodesis: From Preclinical Research to Clinical Scenario

F. Salamanna,1 M. Sartori,2 G. Barbanti Brodano,3 C. Griffoni,3 L. Martini,1

S. Boriani,3 and M. Fini1

1Laboratory of Preclinical and Surgical Studies, Rizzoli Orthopedic Institute, Bologna, Italy2Laboratory of Biocompatibility, Technological Innovations and Advanced Therapies,Rizzoli Research Innovation Technology Department, Rizzoli Orthopedic Institute, Bologna, Italy3Department of Oncological and Degenerative Spine Surgery, Rizzoli Orthopedic Institute, Bologna, Italy

Correspondence should be addressed to M. Sartori; [email protected]

Received 23 September 2016; Accepted 5 January 2017; Published 13 February 2017

Academic Editor: Marco Tatullo

Copyright © 2017 F. Salamanna et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The use of spinal fusion procedures has rapidly augmented over the last decades and although autogenous bone graft is the “goldstandard” for these procedures, alternatives to its use have been investigated over many years. A number of emerging strategies aswell as tissue engineering with mesenchymal stem cells (MSCs) have been planned to enhance spinal fusion rate. This descriptivesystematic literature review summarizes the in vivo studies, dealing with the use ofMSCs in spinal arthrodesis surgery and the stateof the art in clinical applications. The review has yielded promising evidence supporting the use of MSCs as a cell-based therapyin spinal fusion procedures, thus representing a suitable biological approach able to reduce the high cost of osteoinductive factorsas well as the high dose needed to induce bone formation. Nevertheless, despite the fact that MSCs therapy is an interesting andimportant opportunity of research, in this review it was detected that there are still doubts about the optimal cell concentrationand delivery method as well as the ideal implantation techniques and the type of scaffolds for cell delivery. Thus, further inquiry isnecessary to carefully evaluate the clinical safety and efficacy of MSCs use in spine fusion.

1. Introduction

Spinal fusion is a commonmeans to treat vertebral instability.Its use has quickly increased over the last decades in orderto realize the stabilization of the spine in patients affected bydegenerative, oncologic, and traumatic spine diseases. Auto-genous bone harvested from the iliac crests is the standardprocedure for spinal fusion surgery and it is used in morethan 190.000 cases/year in Europe [1]. It owns all the key graftmaterial properties, that is to say osteoconduction, osteoin-duction, osteogenic potential, and also structural integrity ifcorticals are comprised. However, the use of autologous bonegraft has been described to be linked with 5% to 35% non-union rate, intraoperative blood loss, and residual morbidityat the donor sites in about 30% of the patients [2]. Thereare many factors inherent to the spine fusion failure such astensile forces, low bone surface, and interference by sur-rounding musculature [3]. In addition, the time required for

spinal fusion increases with advancing age and the fusion rateremains unpredictable in the ageing population [4]. More-over, smoking, osteoporosis, and systemic illnesses have anadverse impact on bone and in particular in spinal surgery[5, 6].The presence of these intrinsic complications has givenrise to research into newmaterials andmethods avoiding iliaccrest harvesting. Thus, there are differing lines of researchsuch as bone substitutes (allografts, demineralized bonematrix, and ceramics) and osteoinductive growth factors(bone morphogenic proteins). However, bone substitutes,which are merely osteoconductive and not osteoinductive,remain yet to be finished as substitutes for bone because thefusion achieved with them is not solid enough. In fact, for asuccessful spinal fusion to occur, several essential elementsin addition to a biocompatible scaffold are necessary. Theyinclude the presence of the bone-forming cells or theirprecursors and an appropriate biological signal that directbone synthesis. The most critical of these components are

HindawiStem Cells InternationalVolume 2017, Article ID 3537094, 27 pageshttps://doi.org/10.1155/2017/3537094

https://doi.org/10.1155/2017/3537094

2 Stem Cells International

the osteoblasts or their precursors, the mesenchymal stemcell (MSC), both of which own the ability to form bone. Toovercome these limitations, researchers have focused on newtreatments that will allow for safe and successful bone repairand regeneration. In this field, adult stem cells derived frommesenchymal tissue represent a promising source for boneengineering for their ability to differentiate into osteoblasts.MSCs are undifferentiated cells characterized by a highproliferation rate that were found in several adult tissues[7–9]. The multipotent nature of individual MSCs was firstestablished in 1999 by Pittenger et al. [10], and since then theyhave been found to be pluripotent, giving rise to endoderm,ectoderm, and mesoderm cells [11]. Thus, MSCs are wellsuited to therapeutic applications also because they can beeasily cultured and have high ex vivo expansive potential[12–15]. In the treatment of several musculoskeletal injuries,such as bone, articular cartilage, and other joint tissues,MSCsfrom bone marrow (BMSCs) are the most widely used cells,followed byMSCs from adipose tissue (ADSCs) [16–18]. Bothtypes of cells have been demonstrated to have a significanteffect on spinal fusion in a multitude of settings including avariety of culturingmechanisms, scaffolds, and added growthfactors. However,MSCs represent a lesser (0.001–0.01%) frac-tion of the total population of the nucleated cells [19, 20]. Toincrease the concentration of MSCs, several techniques havebeen developed, especially cell ex vivo expansion, but manyproblems limited the clinical application, such as the sterilitytechnique, long culture time, high cost, and the mixtureof human cell culture mediumwith fetal bovine serum.Thus,the method of collecting MSCs, as well as the real number ofMSCs to be transplanted, remains yet to be established.

To date, a great body of research on MSCs for spinalfusion procedures was performed in vitro and in vivo but aclinical customary procedure for the use of cell-based strate-gies for spinal fusion surgery has not been established andcontrasting clinical but also preclinical results were reportedin literature. More importantly, the clinical transferabilityof some protocols is still to be settled, to optimize timeand sources when modified/stimulated cells, custom madescaffolds, and in vitro steps are required [21]. Thus, in thissystematic review, we aimed to evaluate the efficacy of MSCsin spinal arthrodesis procedures considering the preclinicaland clinical studies of the last 10 years to shed light on usingMSCs for spinal fusion treatment.

2. Motivations

2.1. Why a Systematic Review? We have seen the necessity forperforming a descriptive systematic literature review onMSCs use in spinal arthrodesis procedures in order to under-stand if the use of MSCs may represent a valid strategy ableto facilitate and accelerate bone regeneration during spinesurgery providing to researchers and clinicians a beginningpoint with solid foundations allowing this field tomake a leapforward. Our aim is to offer answers to questions such asthe following: “Since bone contains a complex environmentof many cell types, areMSCs able to perform all the necessaryphysiological functions to achieve, facilitate, and acceleratespinal fusion?,” “What happens to MSCs when they are

added to a scaffold?,” “Which source of MSCs is better touse and which techniques (ex vivo expansion and one-stepprocedure) are better to use?,” “How much does the existingpreclinical model reflect the data so far collected in clinicalstudies?,” and “What do we have to do to further clarifythe potential role of MSCs in spinal fusion procedures?”Specifically, we want to summarize the knowledge collectedin nearly 10 years of research, learning from previous preclin-ical and clinical research which used MSCs for spinal fusionprocedures, since there is an exigent need to have successfulspinal fusion.

3. Methods

3.1. Descriptive Systematic Literature Review. Our descriptiveliterature review involved a systematic search that was carriedout, according to the Preferred Reporting Items for Systema-tic Reviews andMeta-Analyses (PRISMA) statement, in threedatabases (https://www.ncbi.nlm.nih.gov/pubmed, https://www.webofknowledge.com, and https://www.scopus.com).In order to evaluate the ongoing clinical studies, the https://www.clinicaltrials.gov website was also checked. The key-words were mesenchymal stromal cells OR mesenchymalstem cells OR mesenchymal/progenitor stromal cells ORmesenchymal/progenitor stem cells AND spinal arthrode-sis OR spinal fusion OR interbody fusion OR vertebralarthrodesis OR vertebral fusion. We sought to identifystudies where MSCs were employed for spinal fusion pro-cedures. Publications from 2006 to 2016 (original articlesin English) were included. A public reference manager(“http://www.mendeley.com”) was used to delete duplicatearticles.

4. Results and Discussion

An initial literature search retrieved 444 references (Figure 1).Hundred and twenty-nine articles were identified usinghttps://www.ncbi.nlm.nih.gov/pubmed, 210 articles wereidentified using https://www.webofknowledge.com, and 105articles were found in https://www.scopus.com. Six addi-tional articles were obtained from the website https://www.clinicaltrials.gov. The resulting references were selected forsupplementary analysis based on the title and abstracts and149 were considered eligible. References were submitted to apublic referencemanager (Mendeley 1.14, “https://www.men-deley.com”) to eliminate duplicate articles. Sixty completearticles were then reviewed to establish whether the publica-tionmet the inclusion criteria and 50 articles were recognizedeligible for the review considering publications from 2006 to2016 (Figures 2(a) and 2(b)).Thirty-nine articles were in vivostudies on small, medium, and large animal models (Tables 1,2, and 3) while the remaining 11 articles were clinical studiesor clinical trials (Tables 4 and 5).

Figure 3 summarizes the main steps of spinal fusion stemcell-based therapy founded in this literature search.

We did not performmeta-analyses of the selected studiesbut reported the results in a descriptive fashion. By consid-ering the studies emerging from this review, we stratified the

https://www.ncbi.nlm.nih.gov/pubmed

https://www.webofknowledge.com


https://www.scopus.com

https://www.clinicaltrials.gov


http://www.mendeley.com/

https://www.ncbi.nlm.nih.gov/pubmed


https://www.scopus.com



https://www.mendeley.com

https://www.mendeley.com

Stem Cells International 3

Table1:Pu

blish

edin

vivo

studies

insm

allanimalmod

elso

nmesenchym

alste

mcells

forspinalarthrod

esisprocedures.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Ovarie

ctom

ized

rat

hPSC

sfrom

adiposetissue

ofpatie

ntsw

ithand

with

out

osteop

orosis

NEL

L-1

DBM

/𝛽-TCP

4weeks

L4-L5

Group1:DBM

/𝛽-TCP

with

hPSC

s(0.25×106cells/m

L)Gr

oup2:DBM

/𝛽-TCP

with

hPSC

s(0.75×106cells/m

L)Gr

oup3:DBM

/𝛽-TCP

with

NEL

L-1

(33.3𝜇

g/mL)

Group4:DBM

/𝛽-TCP

with

NEL

L-1

(66.6𝜇

g/mL)

Group5:DBM

/𝛽-TCP

with

hPSC

s/NEL

L-1atthe

dosage

ofgrou

ps1and

3Gr

oup6:DBM

/𝛽-TCP

with

ahP

SCs/NEL

L-1atthe

dosage

ofgrou

ps2and4

(i)Group

1achievedafusionrateof

20%

(1/5),grou

p2of

28.6%

(2/7),

grou

ps3and4of

20%

(1/5),and

grou

p5of

37.5%

(3/8),andgrou

p6

improved

thefusionratesu

pto

approxim

ately

83.3%

(5/6)

(ii)M

icrocompu

tedtomograph

yim

agingandqu

antifi

catio

nfurther

confi

rmed

solid

bony

fusio

nin

grou

p6

[22]

Rat

Intoto

ratb

one

marrowfro

mfemur

flush

(1.1×107cells/m

L)bF

GF

PEGDA-

co-

A6A

CAhydrogels

(poly(ethylene

glycol)-

diacrylate

hydrogel

(PEG

DA)a

ndN-acryloyl6-

aminocaproic

acid

(A6A

CA))

2,4,6,and8

weeks

L4-L5

Group1:scaffoldwith

bone

marrow

Group2:scaffo

ldwith

bFGF

Group3:scaffoldwith

salin

esolution

(i)Ra

diograph

ssho

wed

fusio

nmassesin4anim

also

utof

7in

each

grou

pat2weeks.A

t4weeks,all

anim

alssho

wed

clear

evidence

ofhard

tissueformation,

with

progressively

increase

at6and8

weeks

(ii)𝜇

-CTim

agingat8weeks

revealed

a51%

ofmineralized

hard

tissuefor

grou

p3,59%

forg

roup

2,and54%

forg

roup

1(iii)Manualp

alpatio

nprovided

evidence

offusio

nin

allgroup

s,with

nosig

nificantd

ifferencesinfusio

nindices

[23]

Rat

Freshbo

nemarrow(BM)cells

(range,0.60to

2.60×106BM

cells)

rhBM

P-2

(0.006

mg/mL)

Absorbable

collagen

spon

ge(A

CS)

8weeks

L4-L5

Group1:2A

CSwith

fresh

BMand

rhBM

P-2

Group2:2A

CSwith

rhBM

P-2

Group3:1A

CSwith

rhBM

P-2

Group4:AC

Swith

BMGr

oup5:AC

Salon

e

(i)In

grou

p1B

Mplus

rhBM

P-2/AC

Ssig

nificantly

increasedthefusionrate

to89%

(16/18)com

paredwith

abase

fusio

nrateof

33%

(4/12

)ingrou

p3

and50%

(6/12

)ingrou

p2(𝑝<0.05)

(ii)N

odifferenceinstr

engthor

stiffn

essw

asdetected

amon

ggrou

p1

andgrou

ps2and3.

(iii)Nofusio

nor

bone

form

ationwas

observed

inther

atso

fgroup

s4and5

[24]


Table1:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rat

Expand

edMSC

s(3×106)from

goatBM

iliac

crest

lentivira

llytransduced

toexpressluciferase

Non

eHA/𝛽-TCP

7weeks

L1-L2and

L4-L5

Group1:no

cells

Group2:MSC

sGr

oup3:MSC

sgam

ma-irr

adiated

(30G

y)Gr

oup4:MSC

sdippedin

liquidN2

(i)Th

eantilu

ciferase

immun

ohistochemistry

show

edno

newlyform

edbo

neor

luciferase-positive

cells.

(ii)H

istologicalstaining

with

Hem

atoxylin/Eosin

high

lighted

nosig

nsof

abon

eformationin

any

grou

ps

[25]

Rat

Expand

edbo

nemarrowfro

mrat

femur

(1×107

cell/mL)

Non

e

Silkfib

roin

(SF)

and

mineralized

silkfib

roin

(mSF)

12weeks

L4-L5

Group1:SF

scaffold

Group2:SF

with

MSC

sGr

oup3:mSF

Group4:mSF

with

MSC

sGr

oup5:autograft

Group6:sham

grou

p

Fusio

nrate,bon

evolum

e,biom

echanicalp

aram

eters,and

histo

logicalscore

show

edno

significantd

ifferencesb

etweengrou

p4andgrou

p5.Group

3was

significantly

greaterfor

most

parametersthangrou

p2

[26]

Rat

Allo

genicM

SCs

Non

e8weeks

L4-L5

Group1:trinity

evolution(D

BMwith

MSC

s)Gr

oup2:graft

on(D

BM)

Group3:DBM

Group4:decorticationon

ly

(i)Fu

sionrateby

radiograph

ywas

8/8forg

roup

1,3/8forg

roup

2,and

5/8forg

roup

3(ii)F

usionrateby𝜇-C

Tandmanual

palpationwas

4/8forg

roup

1,3/8for

grou

p2,and3/8forg

roup

3

[27]


Table1:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Mou

seBo

nemarrow

from

femur

and

tibia(1.0×108

cells/m

L)

PRPfro

mdo

nor(1.0×

109

platelets/mL)

orrhBM

P-2

(31𝜇

g/mL)

ACS

4weeks

L4-L5and

L5-L6

Group1:collagenspon

gewith

rhBM

P-2andsalin

esolution

Group2:collagenspon

gewith

rhBM

P-2andPR

PGr

oup3:collagenspon

gewith

rhBM

P-2andBM


ly

(i)Fu

sionappeared

radiograph

ically

andhisto

logically

similarinallthree

experim

entalgroup

s(ii)Th

earea,volume,anddensity

ofthefusionmassw

eres

ignificantly

greater(𝑝<0.05)for

grou

p3as

comparedwith

grou

p1

(iii)Group

2hadinterm

ediatefusio

narea

anddensity

(iv)N

ospinalfusio

nwas

detected

ingrou

p4

[28]

Rat

Expand

edrat

bone

marrow

from

femurs(1×

106cells/m

L)Fibrin

matrix

PCL-TC

P6weeks

L4-L5

Group1:10𝜇gof

rhBM

P-2with

1×106un

differentiatedBM

SCs

Group2:10𝜇gof

rhBM

P-2with

osteogenic-differentiatedBM

SCs

Group3:2.5𝜇

grhBM

P-2with

undifferentiatedBM

SCs

Group4:2.5𝜇

grhBM

P-2with

osteogenic-differentiatedBM

SCs

Group5:0.5𝜇

grhBM

P-2with

undifferentiatedBM

SCs

Group6:0.5𝜇

grhBM

P-2with

osteogenicdifferentiatedBM

SCs

(i)Predifferentia

tionof

BMSC

sbefore

transplantationfailedto

prom

otep

osterolateralspinalfusion

whencodelivered

with

low-doseo

frhBM

P-2in

grou

p5as

17%

fusio

nratewas

observed

(1/6)

(ii)Incontrast,

combineddeliveryof

undifferentiatedBM

SCsw

ithlow-doseB

MP-2(2.5𝜇g)

asin

grou

p5demon

strated

significantly

high

erfusio

nrate(4/6

or67%)a

swellas

significantly

increasedvolumeo

fnew

bone

form

ation

[29]

Rat

Hum

anbo

nemarrow(5×106

MSC

s)Non

eTitanium

microplates

with

HA

8weeks

L1–L

3Gr

oup1:titanium

microplates

with

HA

Group2:titanium

microplates

with

HA/M

SCs

Histolog

y,histo

morph

ometry,and

𝜇-C

Trevealed

nosig

nificantb

one

form

ationin

grou

p2in

comparis

onwith

grou

p1

[30]


Table1:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rat

ADSC

s(5×106

cells/scaffo

ld)

rhBM

P-2

oradenovira

lvector

containing

BMP-2gene

Type-Icollagen

spon

ge4weeks

L4-L5

Group1:ADSC

stransdu

cedwith

anadenovira

lvectorc

ontaining

rhBM

P-2gene

Group2:ADSC

swith

osteogenic

mediaand1m

g/mLof

recombinant

rhBM

P-2

Group3:rhBM

P-2(10m

g)Gr

oup4:rhBM

P-2(1mg)

Group5:ADSC

s

(i)Allanim

also

fgroup

1were

characteriz

edby

fusio

nmasses(8/8)

after

4weeks

(ii)G

roup

1revealedspinalfusio

nat

thec

ephaladlevel(L3

andL4

)(iii)New

bone

form

ationin

grou

ps1

was

significantly

larger

than

thosein

anyothertreatmentg

roup

(𝑝<0.005)

(iv)G

roup

s3and4show

edas

olid

fusio

nin

8/8and4/8anim

als,

respectiv

ely(v)G

roup

s2and5show

edno

fusio

n

[31]

Rat

hPSC

sfrom

adiposetissue

Non

eDBM

4weeks

L4-L5

Group1:DBM

Group2:DBM

with

0.15×106hP

SCs

Group3:DBM

with

0.50×106hP

SCs

Group4:DBM

with

1.50×106hP

SCs

(i)hP

SCtre

atment(grou

ps2,3,and

4)sig

nificantly

increasedspinal

fusio

nratesincomparis

onwith

grou

p1

(ii)G

roup

s2,3,and

4resultedin

fusio

nrateso

f100%,80%

,and

100%

,respectiv

ely,com

paredwith

20%

fusio

nin

grou

p1

(iii)Com

puteriz

edbiom

echanical

simulation(finiteelem

entanalysis

)furtherd

emon

strated

bone

fusio

nin

hPSC

treatmentg

roup

s(iv

)Histologicalanalyses

show

edendo

chon

dralossifi

catio

nin

hPSC

-treatedsamples

[32]

Rat

ADSC

sfrom

healthydo

nors

(1.0×106)

PurchasedBM

SCs

(1.0×106)

Adenovira

lvectors

adeno-BM

P-2

and

adeno-La

cZused

totransduce

ADSC

sand

BMSC

s

ACS

8weeks

L4-L5

Group1A

CSwith

ADSC

stransfected

with

adeno-BM

P-2

Group2AC

Swith

BMSC

stransfe

cted

with

adeno-BM

P-2

Group3AC

Swith

rhBM

P-2

Group4AC

Swith

ADSC

stransfected

with

adeno-LacZ

Group5AC

Swith

BMSC

stransfe

cted

with

adeno-LacZ

,and

Group6AC

S

(i)Spinalfusio

nwas

observed

ingrou

ps1,2,and3rats

(ii)7

5%(15/20)o

fthe

anim

also

fgrou

psIand

IIhadspon

taneou

sextensionof

thefusionto

asecon

dlevel

(iii)Noanim

alsingrou

ps4,5,and6

ratsdevelopedfusio

n(iv

)New

bone

volumew

assig

nificantly

greateringrou

ps1and

2than

ingrou

p4

[33]


Table1:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rat

Expand

edBM

cells

from

femurs

andtib

ias

(1×106/60𝜇

L)

FGF-4

(41𝜇

g)HA

8weeks

L4-L5

Group1:HA

Group2:HAwith

MSC

sGr

oup3:HAwith

MSC

sand

FGF-4

(i)Ra

diograph

ic,high-resolutio

n𝜇-C

T,andmanualp

alpatio

nrevealed

spinalfusio

nin

5/6(83%

)ingrou

p2

(ii)Ingrou

p1,3/6(60%

)rats

developedfusio

natL4

-L5by

radiograph

yand2/5(40%

)by

manualp

alpatio

nin

radiograph

icexam

ination

(iii)In

grou

p3,bo

nefusio

nwas

observed

inon

ly50%

ofratsby

manualp

alpatio

nandradiograph

icexam

ination

[34]


Table2:Pu

blish

edin

vivo

studies

inmedium

anim

almod

elso

nmesenchym

alste

mcells

forspinalarthrod

esisprocedures.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffoldmaterial

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rabbit

Expand

edBM

from

iliac

crest(1.5

×106cells/m

L)Oste

ogenic

medium

HA

6weeks

L5-L6

Group1:autograft

Group2:HAwith

type

Icollagengel

Group3:HAandtype

Icollagengel

with

MSC

sGr

oup4:HAandtype

Icollagengel

with

MSC

sind

uced

toward

osteogenicph

enotype

Thefusionratesw

ere

4/6in

grou

p1;

0/6in

grou

p2;

2/6in

grou

p3;and

4/5in

grou

p4

[35]

Rabbit

FreshBM

from

iliac

crests

Fibron

ectin

HA

6weeks

L4-L5

Group1:autograft

from

iliac

crest

Group2:autograft

from

transverse

processb

oneg

raft

Group3:HAsticksa

ndiliac

bone

graft

Group4:HAsticksw

ithBM

aspirate

Group5:HAsticks

Group6:HAsticksw

ithFN

andBM

aspirate.


ly

(i)Th

eelasticityandmechanical

strengthweres

ignificantly

high

erin

grou

p1thanin

grou

ps2,4,and5

(ii)Th

emechanicalstre

ngth

achieved

ingrou

ps3and6was

nearlyequalto

thatin

grou

p1

(iii)Th

emechanicalstre

ngth

was

significantly

high

erin

grou

p6than

ingrou

p4

(iv)H

istologyshow

edintraporou

sosteogenesisin

grou

ps3,4,and6

[36]

Rabbit

Expand

edBM

cells

from

iliac

crest(1×

106cells/m

L)

(i)rhBM

P-2

(ii)b

FGF

(iii)Au

tograft

HA

6weeks

L4–L

5

Group1:autograft

Group2:HAwith

MSC

sGr

oup3:HAwith

MSC

sand

BMP

Group4:HAwith

MSC

sand

bFGF

Group5:HAwith

MSC

sand

BMP/bF

GF

Thefusionratesw

ere

4/7in

autograft

grou

p;0/7in

MSC

s/HAgrou

p;2/7in

MSC

s/HA/BMPgrou

p;3/7in

MSC

s/HA/FGFgrou

p;and

6/7in

MSC

s/HA/BMP/bF

GFgrou

p

[37]


Table2:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffoldmaterial

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rabbit

Expand

edBM

cells

from

iliac

crest

Non

eNon

e8weeks

L4-L5

Group1:autograft

Group2:autograft

with

MSC

s(i)

Ingrou

p1,thefusionratewas

53%

(8/15

)(ii)Ingrou

p2,thefusionratewas

0%[38]

Rabbit

BMfro

mfemur,

tibia,trochanter,

andiliac

crest

Non

eTC

P7weeks

L5-L6

Group1:TC

Palon

eGr

oup2:TC

Pwith

MSC

sGr

oup3:TC

Pwith

MSC

sand

LIPU

S

(i)Sign

ificant

increase

inmanual

palpationin

grou

p3tre

ated

with

LIPU

S(86%

)incomparis

onwith

grou

ps1(0%

)and

2(14

%)w

ithou

tLIPU

S(ii)Th

ebon

evolum

eoffusionmass

was

significantly

larger

ingrou

p3

than

theo

ther

twogrou

psby

quantitativec

ompu

tedtomograph

icanalysis

(iii)Group

3fusio

nmassh

adab

etter

osteointegratio

nleng

thbetweenho

stbo

neandim

plantedcompo

sitea

ndpresentedmoren

ewbo

neform

edin

theT

CPim

plants

(iv)G

roup

3hadosteocho

ndral

bridging

,earlystageo

fbon

yfusio

n,fro

mhisto

logicalp

oint

ofview

[2]

Rabbit

Expand

edBM

from

iliac

crest

Non

ePo

ly(la

ctide-co-

glycolide)

(PLG

A)/HA/

type

Icollagen

6weeks

or12

weeks

after

graft

ing

L4-L5

Group1:autograft

Group2:PL

GA/H

A/TypeI

collagen

with

MSC

s

Radiograph

ic,com

putedtomograph

yexam

inations,torsio

nalloading

tests

,andhisto

logice

xaminations

show

edsolid

fusio

nin

3/5rabbits

inbo

thexperim

entalgroup

sat6

weeks

and

5/5solid

fusio

nin

both

grou

psat12

weeks

[39]


Table2:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffoldmaterial

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rabbit

ADSC

sfrom

the

inguinalgroo

veNon

eNano-

hydroxyapatite–

collagen–

polylactic

acid

(nHAC

–PLA

)10

weeks

L5-L6

Group1:autograft

Group2:nH

AC–P

LAGr

oup3:autograft

with

nHAC

–PLA

Group4:ADSC

swith

nHAC

–PLA

(i)Th

erateo

ffusionwas

significantly

high

erin

grou

p1and

grou

p4than

ingrou

p2andgrou

p3

(ii)M

icrostr

ucturalanalysis

ofthe

samples

show

edmoren

ewbo

ne-like

tissueformationin

grou

p1and

grou

p4than

intheo

ther

twogrou

ps(iii)Mechanicalpropertiessho

wed

thatthes

treng

thandstiffn

esso

fgrou

p1and

grou

p4werem

uch

high

erthan

thoseo

fgroup

2and

grou

p3

[40]

Rabbit

BMfro

mfemur

(1.0×108

allogeneicMSC

s)Non

e

Bioresorbable

purifi

edfib

rillar

collagenandcalcium

phosph

atec

eram

ics

containing

HAand𝛽-

TCP

18weeks

L5-L6

Group1:HA/𝛽

-TCP

with

MSC

sGr

oup2:HA/𝛽

-TCP

(i)In

grou

p1C

Tscanning

revealed

excellent

fusio

nin

2/12

rabbits

(17%

),good

fusio

nin

8/12

(66%

),andfair

fusio

nin

2/12

(17%

)(ii)Ingrou

p2ag

oodfusio

nresult

was

foun

din

3/12

rabbits

(25%

),fair

fusio

nin

6/12

(50%

),andpo

orfusio

nin

3/12

(25%

)

[41]

Rabbit

Expand

edhu

man

BMfro

miliac

crest(107)

Non

e

PLGA/BCP

/collagen

graft

and

MSC

/PLG

A/corallin

eHA/collagen

graft

10weeks

L4-L5

PLGA/BCP

/collagenwith

MSC

s(on

theleft

side)

PLGA/corallin

eHA/collagenwith

MSC

s(on

ther

ight

side)

(i)Ra

diograph

ic,C

T,andbo

nemineralcontentanalysessho

wed

continuo

usbo

nebridgesa

ndfusio

nmassincorpo

ratedwith

the

transverse

processes

(ii)B

onem

ineralcontentvaluesw

ere

high

erin

MSC

s/PL

GA/BCP

/collagen

grou

pthan

inMSC

s/PL

GA/corallin

eHA/collagengrou

p

[42]


Table2:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffoldmaterial

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rabbit

Expand

edBM

from

iliac

crest(2

×107)

Bac-BM

P-7

Collagen/TC

P/HA

12weeks

L4-L5

Group1:collagen/TC

P/HA

Group2:collagen/TC

P/HAwith

MSC

sGr

oup3:collagen/TC

P/HA/

Bac-BM

P-7with

MSC

s

(i)In

theC

Tresults,6/12

fused

segm

entswereo

bservedin

grou

p1

(50%

),8/12

ingrou

p2(67%

),and

12/12

ingrou

p3(100%)

(ii)Th

efusionrateby

manual

palpationwas

0%(0/6)ingrou

p1,

0%(0/6)ingrou

p2,and83%

(5/6)in

grou

p3

(iii)Histologyshow

edthatgrou

p3

hadmoren

ewbo

neandmatured

marrowform

ation

[43]

Rabbit

Expand

edand

osteogenic

indu

cedBM

from

iliac

crest

(OMSC

s)

Non

eAC

S8and12

weeks

L4-L5

Group1:AC

Swith

OMSC

sGr

oup2:AC

SGr

oup3:autograft

Group4:no

thing

(i)Bo

nyfusio

nwas

evidentase

arly

as8weeks

ingrou

ps1and

3(ii)A

t8and12

weeks,byCT

and

histo

logica

nalysis

,new

bone

form

ationwas

observed

ingrou

ps1

and3andfib

rous

tissuea

ndabsence

ofnewbo

newerep

resent

ingrou

ps2

and4

(iii)Manualp

alpatio

nshow

edbo

nyfusio

nin

40%

(4/10

)ofrabbitsin

grou

p1,70%

(7/10

)ofrabbitsin

grou

p3,and0%

(0/10

)ofrabbitsin

both

grou

ps2and4

[44]

Rabbit

Expand

edBM

from

iliac

crest

(105)

MSC

stransduced

with

Smad1C

gene

Absorbablegelatin

spon

ge4weeks

L6-L7

Group1:BM

SCstransdu

cedwith

Smad1cwith

Ad5vector

Group2:BM

SCstransdu

cedwith

Smad1cwith

Ad5vector

retargeted

to𝛼Vintegrins(RG

D)

Group3:BM

SCstransdu

cedwith

BMP-2with

Ad5vector

Group4:BM

SCstransdu

cedwith

BMP-2with

Ad5vector

retargeted

to𝛼Vintegrins(RG

D)

Group5:BM

SCstransdu

cedwith

anAd

5vector

expressin

gb-galactosidase

(i)Th

eareao

fnew

bone

form

edin

grou

ps1,2,3,and4was

significantly

greaterthanthea

reao

fnew

bone

form

edin

grou

p5(p<0.04

fore

ach

grou

pcomparedwith

grou

p5)

(ii)G

roup

4mediatedag

reater

amou

ntof

newbo

neform

ationthan

grou

p3

(iii)Similarly

,group

2mediateda

greatera

mou

ntof

newbo

neform

ationthan

grou

p1(𝑝<0.0007)

(iv)G

roup

2mediatedag

reater

amou

ntof

newbo

neform

ationthan

theo

ther

grou

ps(𝑝<0.02)

[45]


Table2:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffoldmaterial

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rabbit

Expand

edand

osteogenic

indu

cedBM

from

iliac

crest(2×106)

rhBM

P-2

Alginates

caffo

ld16

weeks

L4-L5

Group1:autograft

Group2:alginatescaffoldwith

MSC

sGr

oup3:alginatescaffoldwith

MSC

sandrhBM

P-2


rhBM

P-2

(i)Ra

diograph

icun

ionof

grou

p1

was

11/12

,ofgroup

28/11,ofgroup

311/12

,and

ofgrou

p40/12

(ii)M

anualp

alpatio

nhigh

lighted

6/6

solid

fusio

nin

grou

p1,1/6

ingrou

p2,

5/6in

grou

p3,and0/6in

grou

p4

(iii)Th

emechanicalanalysis

(failu

retorque)d

idno

tdiffer

significantly

betweengrou

p1and

grou

p3that

wereb

othhigh

erthan

grou

p2

[46]

Rabbit

Expand

edand

osteogenic

indu

cedBM

from

iliac

crest(2×106)

Non

eAlginates

caffo

ld12

weeks

L4-L5

Group1:alginatescaffold


MSC

sGr

oup3:alginatescaffold/

hyperbaric

oxygen

(HBO

)therapy

with

MSC

s

Radiograph

icexam

inationand

manualp

alpatio

nhigh

lighted

noun

ionforg

roup

1(0/12),10/22for

grou

p2,and6/12

forg

roup

3[47]

Rabbit

Expand

edBM

from

iliac

crest

TCP

Recombinant

baculovirus

encoding

BMP-2

(Bac-C

B)and

vascular

endo

thelial

grow

thfactor

(Bac-VEG

F)

12weeks

L4-L5

Group1:TC

PGr

oup2:TC

Pwith

MSC

Group3:TC

Pwith

MSC

s/Ba

c

(i)Ra

diograph

icallyfusio

nratewas

detected

asbeing0/12

ingrou

p1,4/12

ingrou

p2,and10/12

ingrou

p3

(ii)M

anualp

alpatio

nhigh

lighted

nofusio

nsin

grou

p1,twosolid

fusio

nsin

grou

p2,andfives

olid

fusio

nsin

grou

p3

[48]


Table2:Con

tinued.

Animalmod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffoldmaterial

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Rabbit

Expand

edand

osteogenic

indu

cedBM

from

iliac

crest

Bioresorbable

hydrogel

(pluronicF

27)

andcoralline

HA

Non

e6and12

weeks

L4-L5

Group1:Pluron

ic127/HAhybrid

graft

with

MSC

sGr

oup2:autograft

(i)Solid

fusio

nwas

achieved

in3/5

rabbits

from

both

grou

p1and

2at6

weeks,and

solid

fusio

nwas

present

in5/5fro

mbo

thgrou

pat12

weeks

(ii)N

odifferences

wered

etected

betweenthetwogrou

psfor

biom

echanicalanalysis

andfro

mhisto

logicalp

oint

ofview

[49]


Table3:Pu

blish

edin

vivo

studies

inlargea

nimalmod

elso

nmesenchym

alste

mcells

forspinalarthrod

esisprocedures.

Animal

mod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Pig

ADSC

sfrom

ingu

inal

subcutaneous

tissue

Non

eDBM

8and12

weeks

L2–L

6

Group1:on

ecagew

asleftandthree

filledwith

freezed

riedirr

adiated

cancellous

pigbo

negraft

Group2:fre

ezed

riedirr

adiated

cancellous

pigbo

negraft

Group3:cancellous

bone

autograft

Group4:bo

negraft

with

3DosteogenicdifferentiatedADSC

s

𝜇-C

Tscan,m

icroradiograph

y,andhisto

logy/histom

orph

ometry

demon

strated

asignificant

increase

inbo

necontentingrou

p4

[50]

Sheep

Expand

edand

osteogenic

indu

cedBM

SCs

from

iliac

crest

(5-6×107)

Fibrin

TCP/HA

12weeks

L1–L

6

Group1:HAwith

MSC

sGr

oup2:TC

P/HAwith

MSC

sGr

oup3:autograft

(i)Ra

diograph

y,manual

palpation,

histo

logicalanalysis

,andSE

Manalyses

revealed

demon

strated

bette

rbon

eform

ationin

grou

p2compared

togrou

p1

(ii)H

istom

orph

ometry

detected

55.8%of

newbo

nein

grou

p3,

follo

wed

bygrou

p2(42.7%

)and

grou

p1(10.7%)

[51]

Sheep

Allo

genics

heep

mesenchym

alprecursorc

ells

(MPC

s)fro

mBM

from

iliac

crest

Non

eHA/TCP

16–36weeks

L2–L

5

Group1:autograft

Group2:HA/TCP

Group3:HA/TCP

with

MPC

s(25×

106)

Group4:HA/TCP

with

MPC

s(75×

106)

Group5:HA/TCP

with

MPC

s(225

×106)

Com

putedtomograph

y,high

-resolutionradiograph

y,biom

echanicaltestin

g,organ

pathology,bo

nehisto

pathology,

andbo

nehisto

morph

ometry

show

edthatallogeneic

mesenchym

alprecursorc

ells

prod

uced

fusio

neffi

cacy

similar

tothatachieved

usingiliac

crest

autograft

[52]


Table3:Con

tinued.

Animal

mod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Sheep

Allo

genicM

PCs

from

BMfro

msheepiliac

crest

Non

eHA/TCP

16weeks

L4-L5

Group1:autograft

Group2:HA/TCP

with

MPC

s(2.5×

106)

Group3:HA/TCP

with

MPC

s(6.5×

106)

Group4:HA/TCP

with

MPC

s(12.5

×106)

(i)Manualp

alpatio

nof

the

fusio

nsiteind

icated

solid

fusio

nin

morethan75%of

MPC

-treatedgrou

pand65%of

grou

p1

(ii)C

ompu

tedtomograph

yand

histo

morph

ometry

analyses

show

edallanimalsintheM

PCs

grou

psandgrou

p1fusionmasses

werep

resent

at16

weeks

[53]

Sheep

Expand

edand

osteoind

uced

BMfro

miliac

crest

Non

eHA

6mon

ths

L4-L5

Group1:autograft

Group2:allograft

Group3:HA

Group4:HAwith

MSC

s.

(i)By

CTscan

andhisto

logy

lumbarfusionwe

rehigh

erfor

grou

ps1and

2(70%

)thanfor

grou

p3(22%

)and

grou

p4(35%

)(ii)N

ewbo

neform

ationwas

high

erforg

roup

s1and2

(iii)Group

4hadab

etterfusion

ratethan

grou

p3,bu

tthe

histo

logy

show

edno

significant

differences

betweenthem

interm

sofq

uantity

ofbo

neform

ation

[54]

Sheep

BMconcentrate

(1.5×106in

0.2

mL)

Non

e

Naturalbo

necollagenscaffold

(NBC

S)fro

mhu

man

organic

bone

particles

6and10

weeks

L3-L4and

L4-L5

Group1:autograft

Group2:NBC

SGr

oup3:BM

CsGr

oup4:NBC

Swith

BMCs

(i)Solid

spinalfusio

nwas

achieved

inallsixsegm

ents(6/6)

ingrou

p4at10

weeks,com

pared

with

4/8segm

entsin

grou

p1,2/8

segm

entsin

grou

p2,and3/6

segm

entsin

grou

p3

(ii)Th

ebiomechanicalstiff

ness

offusio

nmassesa

ndbo

nevolumea

tthe

fusio

nsitew

ere

high

erin

grou

p4(p<0.05)

(iii)At

10weeks,the

radiograph

icscorer

eached

was

significantly

high

erin

grou

p4

than

ingrou

ps1,2and3

(iv)H

istologicalfin

ding

srevealed

thatgrou

p4indu

ced

newbo

neform

ationintegrated

wellw

ithho

stbo

netissue

[55]


Table3:Con

tinued.

Animal

mod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Ewes

Allo

genicM

PCs

(5×106)

orallogenic

amnion

epith

elialstem

cells

(5×106AEC

s)

Non

e

Fidjiinterbo

dycage

madefrom

polyetherether-

ketone

andHA/TCP

3mon

ths

C3-C

4

Group1:cage

packed

with

autograft

Group2:cage

packed

with

HA/TCP

Group3:cage

packed

with

HA/TCP

andMPC

sGr

oup4:cage

packed

with

HA/TCP

andAEC

sGr

oup5:controls

(i)Sign

ificant

fusio

nmassw

asdetected

ingrou

p3comparedto

thatin

grou

ps1,2,or

4(ii)C

Tscan

at3mon

thsrevealed

that5/6anim

alsingrou

p3(83%

)hadcontinuo

usbo

nybridging

comparedwith

0/5of

grou

p4

and1/6

ofgrou

p1and

2/6of

grou

p2(p<0.01)

[56]

Ewes

Allo

geneic

MPC

s(5×106or

10×

106)

Non

e

Fidjiinterbo

dycage

madefrom

polyetherether-

ketone

andHA/TCP

3mon

ths

C3-C

4anterio

rcervical

discectomy

andfusio

nwith

ainterbod

ycage

Group1:cage

packed

with

autograft

Group2:cage

packed

with

HA/TCP

Group3:cage

packed

with

HA/TCP

and5×106MPC

sGr

oup4:cage

packed

with

HA/TCP

and10×106MPC

sGr

oup5:controls

(i)Nosig

nificantd

ifferences

werefou

ndbetweengrou

ps3

and4

(ii)C

Tscan

show

edthat9/12

(75%

)MPC

-treatedanim

alsh

adcontinuo

usbo

nybridging

comparedwith

1/6of

grou

p1and

2/6of

grou

p2(p<0.019andp<

0.04

4,resp.)

(iii)By

quantitativeC

T,density

ofnewbo

nein

MPC

-treated

anim

alsw

as121%

high

erthan

ingrou

p2(p<0.017)

and128%

high

erthan

ingrou

p1(p<

0.00

01)

[57]

Pig

BMSC

s(10×106)

rhBM

P-2

(0.6mg)

Bioresorbable

scaffolds

made

from

medical

gradep

oly

(Σ-

caprolactone)-

20%tricalcium

phosph

ate

(mPC

L/TC

P)

9mon

ths

L2-L3and

L4-L5

Group1:mPC

L/TC

Pwith

rhBM

P-2

Group2:mPC

L/TC

Pwith

BMSC

sGr

oup3:mPC

L/TC

PGr

oup4:autograft

(i)Th

emeanradiograph

icscores

were3

.0,1.7,

1.0,and

1.8for

grou

ps1to4,respectiv

ely(ii)Th

ebon

evolum

efractionof

grou

p1w

astwofoldhigh

erthan

grou

p2

(iii)Histolog

y,𝜇-C

T,and

biom

echanicalevaluation

show

edsolid

andcomparable

fusio

nbetweengrou

ps1and

4(iv

)Group

2show

edinferio

rqu

ality

offusio

nwhencompared

with

grou

ps1and

4whilegrou

p3show

edno

fusio

neven

at9

mon

ths

[58]


Table3:Con

tinued.

Animal

mod

elMSC

ssou

rce

Other

biological

adjuvant

Scaffold

material

Experim

ental

time(

weeks)

Spinalfusio

nlevel

Experim

entald

esign

Mainou

tcom

eRe

ference

Ovine

Autogeno

uswho

leBM

orBM

concentrate

Non

eTC

P6mon

ths

L4-L5

Group1:autograft

Group2:TC

Pwith

BMconcentrate

Group3:TC

Pwith

who

lebo

nemarrow/

Group4:TC

P.

(i)At

6mon

ths,33%of

grou

p2

and25%of

theg

roup

1sitesw

ere

fused,comparedwith

8%of

grou

p3and0%

ofgrou

p4

(ii)H

istolog

yof

fusedsamples

show

eddenser

bone

form

ation

ingrou

p2than

ingrou

p1sites

[59]


Table4:Pu

blish

edclinicalstudies

involvingtheu

seof

mesenchym

alste

mcells

forspinalarthrod

esisprocedures.

Arthrod

esislevel

MSC

ssou

rce

Cell

manipulation

Treatm

ent

Patie

nt’snu

mber

(meanage)

Follo

w-up

Com

plications

Reference

Sing

lelevel=

222or

morelevels

=13

Right

poste

rosuperio

riliac

crest

Freshbo

nemarrow

(i)Leftsid

e:autologous

bone

graft

(ii)R

ight

side:mixture

ofBC

Pand

fresh

autogeno

usbo

nemarrow

35(24males,11

females)

Meanage=

59.2

Minim

um30

mon

ths

1pseud

oarthrosis

[60]

Sing

lelevel=

142levels=23

3levels=4

Rightand

left

iliac

crest

Bone

marrow

concentrate

(enrichedusinga

cellseparator)

(i)Decom

pressio

ncases:locally

harvestedbo

necombinedwith

autologous

enric

hed

MSC

s/𝛽-TCP

(ii)N

ondecompressio

ncases:

autologous

enric

hedMSC

s/𝛽-TCP

41(30men,11

wom

en)

Meanage=

44.0

Median36.5

mon

ths

(i)4patie

ntsw

ithtransie

ntexud

ationor

mod

erates

wellingin

theirw

ound

s(ii)2

pseudo

arthrosis

(iii)1p

atient

with

bursas

ynovialis

(iv)1

patie

ntwith

progressive

insta

bilityof

thea

djoined

supra-vertebra

[20]

1and

2levels

Poste

rior

iliac

crest

Bone

marrow

concentrate

(i)Side

1:concentrated

bone

marrowassociated

with

macropo

rous

biph

asiccalcium

phosph

atec

eram

icsg

raftand

autologous

bone

(ii)S

ide2

:non

concentrated

bone

marrowwith

ceramicsg

raftand

autologous

bone

15Meanage=

46.3

24mon

ths

Non

e[61]

1,2,or

3levels

One

iliac

crest

Bone

marrow

concentrated

(i)Side

1:allograft

plus

autologous

bone

marrowconcentrate

(ii)S

ide2

:autologou

siliacc

rest

bone

25(15males

and10

females)

Meanage=

45.6

24mon

ths

Non

e[62]

Not

specified

Poste

rioriliac

crests

Bone

marrow

concentrate

(i)40

patie

nts:allograft

chipsa

lone

(ii)4

0patie

nts:spon

giou

sallo

graft

chipsm

ixed

with

bone

marrow

concentrate

80(22men,58

females)

24mon

ths

Twocomplications

occurred

ineach

ofthetwo

grou

ps:hem

atom

awith

subsequent

revisio

nsurgeryand

drainage

durin

gthefi

rstw

eek

posto

perativ

ely

[63]

Not

specified

Sing

leiliac

crest

Bone

marrow

concentrate

31patie

nts:concentrated

bone

marrowaspiratewith

allograft

and

demineralized

bone

matrix

31(9

men

and22

females)

Meanage:71.5

Atleast12

mon

ths

(i)One

seroma

(ii)O

nepseudarthrosis

(iii)Th

reer

eoperatio

nfor3

patie

nts

fora

djacentsegmentp

atho

logy

[64]

1or2

levels

Non

applicable

Allo

graft

cellu

lar

bone

matrix

containing

nativ

emesenchym

alste

mcells

and

osteop

rogenitor

cells

182patie

nts:allograft

cellu

larb

one

matrix

containing

nativ

emesenchym

alste

mcells

and

osteop

rogenitorc

ells

182

(49%

female,

51%male)

Meanage:51

24mon

ths

(i)1d

urotom

y(ii)2

wou

ndinfections

(iii)2incidences

ofnew

radiculopathy

(iv)1

incidenceo

fhypotensio

n(v)1

incidenceo

fhypertension

(vi)2incidences

ofpo

stoperativ

esoft-tissues

wellin

g

[65]


Table5:Listof

clinicaltria

lsinvolvingmesenchym

alste

mcells

forspinalarthrod

esisprocedures

(from

clinicaltrials.gov).

ClinicalTrials.gov

Identifi

erCon

ditio

nStud

ytype

Estim

ated

enrollm

ent/

enrolledpatie

nts

MSC

data

(sou

rce,manipulation,

orstr

ategy)

Num

bero

fcells

Stud

yarms

Follo

w-up

(mon

ths)

Activ

ity

NCT

01552707

Degenerative

spon

dylolisthesis

grades

I-II

Interventio

nal

phases1-2

62

Expand

edautologous

mesenchym

alste

mcells

obtained

underG

MPcond

ition

sfixedin

allogenicb

onetissue

Not

repo

rted

(i)Group

1:instr

umented

spinalfusio

nandthetissue

engineeringprod

uct

compo

sedby

“exvivo”

expand

edautologous

mesenchym

alste

mcells

fixed

inallogenicb

one

tissueinspinalfusio

n(ii)G

roup

2:sta

ndard

treatmento

finstrum

ented

spinalfusio

nandpatie

nt's

bone

iliac

crest

12mon

ths

Recruitin

g

NCT

00549913

Poste

rolateral

lumbarfusion

Interventio

nal

phases1-2

42

Immun

osele

cted,

cultu

re-expanded,nu

cleated,

allogeneicmesenchym

alprogenito

rcells

Not

repo

rted

(i)Ex

perim

entalgroup

1:lowestd

oseo

fNeoFu

se(ii)E

xperim

entalgroup

2:middled

oseo

fNeoFu

se(iii)Ex

perim

entalgroup

3:high

estd

oseo

fNeoFu

se(M

PCs)

(iv)C

ontro

lgroup

:autologous

bone

graft

24and36

mon

ths

Com

pleted

NCT

01513694

Intervertebraldisc

disease

Interventio

nal

phases1-2

15

Cellsuspensionof

MSC

sfrom

bone

marrowaspirateexpand

edin

vitro

inas

pecific

medium

enric

hedwith

plateletlysate

with

outadd

ition

ofanim

alprod

ucts

Not

repo

rted

(i)Au

tologous

mesenchym

alste

mcells

arrang

edin

apho

sphate

ceramic

Not

repo

rted

Unk

nown

NCT

01603836

Spon

dyloarthrosis

,spon

dylosis

Interventio

nal

80Spon

giou

sallo

graft

chipsm

ixed

with

bone

marrowconcentrate

74×104/L

ataverage(range,

1.06–

1.98×

104/L)

(i)Group

1:spon

giou

sallograft

chipsa

lone

(ii)G

roup

2:spon

giou

sallograft

chipsm

ixed

with

bone

marrowconcentrate

24mon

ths

Com

pleted

https://www.clinicaltrials.gov/

https://clinicaltrials.gov/ct2/show/NCT01552707





Articles located through learning-basedapproach

PubMed, n = 129 Web of Science, n = 210 Scopus, n = 105

Potentially relevant articles identified andtitle/abstract screened (n = 450)

Articles excluded (n = 301):(i) Noninherent and/or on other spinal pathology (i.e., spinal cord injury,

intervertebral disc regeneration, nucleus pulposus, spina bifida) (n = 213)(ii) Non-English article (n = 4)

(iii) In vitro studies (n = 10)(iv) Nonoriginal articles, reviews (n = 62)(v) Only abstract with data unabstractable (n = 10)

(vi) Withdrawn and noninherent clinical trials (n = 2)

Results sent to “Mendeley” to delete duplicatearticles (n = 60)

Potentially relevant articles for the review(n = 149)

Articles excluded on basis of full text (n = 10):(i) Case report (n = 1)

(ii) Noninherent in vivo studies (n = 7)(iii) Withdrawn clinical trials (n = 1)(iv) Noninherent clinical trial (n = 1)

Relevant articles identified on basis of full text(n = 50)

In vivo studies (n = 39) Clinical studies (n = 7)

Articles obtained from other sources:https://www.clinicaltrials.gov (n = 6)

Clinical trials (n = 4)

Figure 1: Systematic literature review flow diagram. Flow of information through the different phases of the systematic review.

papers according to in vivo studies (small, medium, and largeanimal models) and clinical trials.

4.1. In Vivo Studies

4.1.1. Small Animal Models. Thirteen studies (Table 1)employed MSCs in small animal models (𝑛 = 1 in mice and𝑛 = 12 in rats) in order to achieve or improve spinal fusionrate. In the majority of the studies the spinal fusion surgery

was carried out by decortications of L4 and L5 but also L1-L2 [25] and L4–L6 [28] transverse processes. Klıma et al.[30] used titaniummicroplates and titanium screws to fix thespinous processes of L1–L3 vertebrae. The experimental timeafter surgery ranges from 4 to 8 weeks. Most of the studiesused in vitro expanded MSCs [22, 25–27, 29–34] principallyderived from bone marrow [25–27, 29, 30, 33, 34] but alsofrom adipose tissue [22, 31–33]. Beyond the use of expandedMSCs, some authors, in order to take advantage not only


Publications

2006

2007

2008

2009

2010

2011

2012

2013

2014

2015

2016

Years

0123456789

Num

ber o

f in

vivo

stud

ies

(a)

2006

2008

2010

2012

2014

2016

2015

2013

2011

2009

2007

YearsPublications

0123456789

Num

ber o

f clin

ical

stud

ies

(b)

Figure 2: Historical distribution of (a) in vivo models and (b) clinical studies on MSCs use in spinal arthrodesis procedures according to theyear of publication.

Stem cell isolation

Bone marrow in toto

MarrowSpongy bone

Adipocyte

Erythrocyte

Normoblast

MyeloplaxMyelocyte

Myelocyte

Myelocyte

Normoblast withdividing nucleus

Bone marrow

Eosinophil Spinal arthrodesissurgery

Adipose tissue

Adipose derived MSCs ✓Expanded✓Differentiated in osteogenic medium

Combination ortotoinmarrowboneofMSCs derived from bone marrow or

adipose tissue with a scaffold

✓Expanded (differentiated and undifferentiated)✓Transfected✓Codelivered with growth factor or osteogenic protein

Alizarin red staining

ALP staining+

Figure 3: Flow chart summarizing the main steps of spinal fusion procedure when stem cell therapy is used.

by the mesenchymal component but also by the presence oftrophic factors, cytokines, and extracellular matrix molecule,used bone marrow in toto [23, 24, 28]. Few studies usedautologous MSCs [23, 29, 34] while the majority employedallogenic MSCs [22, 24–26, 30–33]. All the examined studiesinvolved seeding cells into allograft [22, 27, 32] or into variousscaffolds, such as ceramic [25, 30, 34], collagen sponge [24, 28,31, 33], silk fibroin [26], and composites [23, 29]. Only oneauthor employed autologous bone graft as control material[26] while the others used allografts or scaffolds withoutMSCs as control. When expanded MSCs were used the cellnumber, loaded on allografts or scaffolds, is 1.0–1.50× 106 [26,29, 32–34] but also lower concentration as in the study of Leeet al. (0.25 × 106) [22] or higher concentration was used [23,26, 30, 31]. Differently from the studies where bonemarrow intoto [23, 24, 28] and undifferentiatedMSCswere cultured andloaded on a scaffold [25, 26, 30, 32–34] four studies [22, 29,

31, 34] employed cells cultured in osteogenic differentiationmedium. In particular, Rao et al. examined also the role of lowdoses bone morphogenic protein- (BMP-) 2 codelivered withboth undifferentiated and differentiated BMSCs showing thatundifferentiated BMSCs with low-dose BMP-2 loaded ona composite scaffold demonstrated superior fusion rate incomparison to all the other examined groups. Low dose ofBMP-2 was also evaluated in association with bone marrowin toto loaded on a collagen sponge, showing that freshbone marrow aspirate increases the osteogenic potency andbiologic efficiency of BMP-2 [24, 28] also in comparison toBMP-2 associated with other adjuvant factors such as plateletrich plasma [28]. BMP-2was used also byMiyazaki et al. in anathymic rat model to compare the efficacy of human ADSCsand BMSCs transduced with an adenovirus containing thecDNA for BMP-2 loaded on collagen sponge.Authors showedthat ADSCs transfected with adeno-BMP-2 induce abundant


bone formation in a manner similar to genetically modifiedBMSCs [33]. Similar results were also obtained by Hsu et al.[31] that demonstrate the potential of adipose derived stemcell as cellular vehicle for this osteoinductive factor. Contraryto the positive effect on spinal fusion derived by the associa-tion of MSCs or bone marrow with BMP-2, the associationof fibroblast growth factor-4 (FGF-4) with differentiatedBMSCs loaded on a HA scaffold did not stimulate fusionbut appears to induce fibrotic change rather than differen-tiation to bone [34]. Despite these negative results, recentlyShih et al. [23] suggested a good performance in promotingspinal fusion rate associating new biomineralized matriceswith bone marrow or basic FGF. Differently from the above-mentioned studies that used growth factors in associa-tion with BMSCs to enhance spinal fusion, other authorsdetermined the efficacy of a 𝛽-tricalcium phosphate (TCP)/demineralized bone matrix (DBM) [22] or DBM alone [32]loaded with different doses of human perivascular stem cells(hPSCs) also in presence and absence of osteogenic proteinNELL-1. Authors highlighted that both in healthy [32] and inosteoporotic condition [22] the presence of hPSCs [32] also inassociation with NELL-1 significantly improved spinal fusion[22]. Differently from all the other studies, Klıma et al. [30]adopted an instrumentedmodel of interspinous fusion show-ing a nonsignificant new bone formation in animals treatedwith hydroxyapatite (HA) and BMSCs in comparison to ani-mals treatedwith scaffold alone, even if in presence of BMSCsauthors describedminor inflammatory reaction compared tothe animals treated without BMSCs. Finally, since the fateand contribution of the MSCs are not sufficiently clarified,especially at clinically relevant locations, Geuze et al. [25]using the bioluminescence imaging of luciferase-markedMSCs and adopting different experimental setup tried toelucidate and clarify the contributionmade not only byMSCsitself on spinal fusion but also by the paracrine effect ofMSCswhen loaded on a ceramic scaffold. Results suggested that thesoluble factors or the presence of extracellular matrix was notsufficient to induce bone formation; thus unfortunately theydid not provide an answer to the critical question whetherthe principal mechanism of action of MSCs is based on theiractivity on the release of soluble mediators.

4.1.2. Medium Animal Model. MSCs treatment to achievespinal fusion was employed in 16 in vivo studies that usedmedium sized animal models (Table 2). All the studies useda single level posterolateral transverse process arthrodesisbetween L4-L5 or L5-L6 or L6-L7. With the exception of onestudy [44] spinal fusion surgery was carried out by creatinga defect between L4 and L5 (depth of 5mm and diameter of10mm); in all the others studies a transverse process decor-tication was performed. The experimental time after surgeryranges from 4 up to 18 weeks. Unless for the study by Kogaet al. [36] that use fresh bone marrow to enhance the spinalfusion rate all the other authors used expanded, autologous[2, 35, 37–40, 42–47, 49], or allogeneic [41, 48] MSCs isolatedfrom bonemarrow [2, 35, 37, 38, 41–49] or adipose tissue [40]at different dosages (from 1.0 × 106 cells to 1.0 × 108). Someauthors used MSCs with osteogenic differentiation [35, 37–40, 42, 44–47, 49] to increase the fusion rate while other

used undifferentiated MSCs [2, 36, 41, 43, 48]. In all thestudies MSCs were loaded on a scaffold (i.e., ceramic,polymeric, collagen sponge, and gelatin sponge) with theexception of Urrutia et al. [38] that used a pellet of culturedBMSCs cografted with an autologous bone graft and showedthat adding differentiated BMSCs in a pellet without ascaffold not only failed to increase fusion rate, but completelyinhibited bony growth. Differently, Nakajima et al. [35], usingdifferentiated BMSCs plusHA, obtained a high rate of lumbarfusion similar to that obtained using autograft alone. In mostof the studies the experimental treatment with MSCs wascompared with autologous bone but in some of them thiscomparison is missing [2, 41–43, 45, 47, 48]. Niu et al. [42]compared BMSCs cultured in a biphasic calcium phosphatewith BMSCs cultured with coralline HA. Coralline HA wasused also by Chen et al. [49] who used MSCs fluorescentlabeledwith PKH-67 dye in combinationwith a bioresorbablehydrogel and coralline HA in comparison to autograft show-ing similar results between groups. In addition, to the use of aceramic graft in associationwithBMSCs, several authors usedBMP to enhance the osteogenic potency of MSCs [37, 43, 46]showing that MSCs in combination with BMP-2 enhancedbone formation in posterolateral spine fusion exerting amore osteoinductive action than MSCs alone [46]. Favorableresults were also obtained comparing the association of acomposite [43] and a ceramic [48] material with baculovirusgenetically modified BMSCs overexpressing BMP-7 [43] orBMP-2 associated vascular endothelial growth factor (VEGF)[48] with nongenetically modified BMSCs. Additionally,Minamide et al. [37] tested also the hypothesis that bothBMP-2 and basic fibroblast growth factor (FGF) mutuallyacted on the proliferation and osteogenic differentiation ofrabbit BMSCs. They showed that the combined treatmentwith BMP-2 and basic FGF produced a favorable degree ofspinal fusion comparable to autograft. An increased spinalfusion rate was also obtained by Koga et al. [36] assessingthe osteogenic potential of HA sticks soaked with freshbone marrow and fibronectin (FN). Interesting were also theresults obtained byHui et al. [2] that underlined that the com-bination of synthetic biomaterials, autologous differentiatedBMSCs, and also low-intensity pulsed ultrasound promotespinal fusion. Differently from the use of low-intensity pulsedultrasound, the use of hyperbaric oxygen therapy adminis-trated to the animals did not enhance the spinal fusion ratewhen a combination of allogenic differentiate MSCs/alginatescaffold was evaluated [47]. The effectiveness of autologousdifferentiated BMSCs was evaluated also by Yang et al. [44]in association with a collagen sponge showing a high fusionrate similar to autologous bone. Another approach exploitedby Douglas et al. is the ex vivo transfer of a gene encodingan osteoinductive factor to BMSCs which are subsequentlyreimplanted into the host. In this study Smad1C gene wastransferred into rabbitMSCs isolated from bonemarrow.Therationale for the use of this approach is to control more effi-ciently bone formationmimicking the natural cascade signalsand avoiding the drawbacks associated with the direct useof BMPs. Authors showed that animals BMSCs transducedex vivo with the Smad1C-expressing tropism-modified Ad5vector mediated a greater amount of new bone formation


than BMSCs transduced with any other vector [45]. Differ-ently from all the other studies Urrutia et al. [38] evaluating acomposite of hot compression-molded PLGA, HA, and type Icollagen as anBMSCs carrier for a posterolateral spinal fusionused also a PKHfluorescence labeling system and highlightedthat the transplanted BMSCs were partly responsible for thenew bone formation. Positive results were also obtained usingallogeneic undifferentiated rabbit BMSCs added to a type Icollagen and calciumphosphate ceramics that promote spinalfusion and did not induce an adverse immune response [41].Only one study evaluated the effectiveness of autologousADSCs combined with a new mineralized collagen matrix(nHAC–PLA) for posterolateral spinal fusion. Results indi-cated that the rate of fusion was significantly higher in theautologous bone and ADSCs + nHAC-PLA groups thanthat in the nHAC-PLA and autologous bone + nHAC-PLAgroups, demonstrating the effective impact of the scaffold alsowhen combined with ADSCs [40].

4.1.3. Large Animal Models. Ten studies used ovine as largeanimal models (Table 3) [51–57, 59] while two used swine[50, 58].These studies carried out four-level, three-level, two-level, or single level spinal fusions surgery all instrumentedwith screws and bars with the exception of Gupta et al. thatused a single level noninstrumented lumbar fusion [59]. Inthese studies both autologous bone marrow or expandedMSCs [50, 51, 54, 55, 58] and allogeneic bone marrow ormesenchymal precursor cells [52, 53, 56, 57, 59] were used toenhance spinal fusion and all of them involved seeding cellsinto allografts [50], collagen scaffolds [55], ceramics [51–54,56, 57, 59], and composites [58] scaffolds. Except for Schubertet al. who use MSCs derived from adipose tissue [50] andGoldschlager et al. that used amnion epithelial cells [57] allthe other authors used differentiatedMSCs andmesenchymalprecursor cells derived from bone marrow but also bonemarrow in toto loaded on the scaffolds at different dosages.Wheeler et al. also compared different dosages of MSCs [52,53, 56]. All the researchers used autografts and grafts withoutcells as controls and the majority highlighted superior resultof the graft associated with MSCs and bone marrow incomparison to the graft alone, while similar [50] or best [51–53, 55, 56, 59] results were seen for autografts in comparisonto grafts associated with MSCs. In addition, Goldschlager etal. showed superior results of mesenchymal precursor cellsloaded onMastergraftmaterial also in comparison to amnionepithelial cells [57]. Differently from the above-mentionedstudies, Cuenca-Lopez et al. observed that bone autograftsperformed better than MSCs loaded on hybrid constructs[54]. Inferior results were also observed for MSCs loaded ona composite scaffold in comparison to autograft but also incomparison to scaffold associated with BMP-2 [58].

4.2. Clinical Studies. The search strategy identified 10 clinicalstudies (Table 4) about MSCs used for spinal fusion proce-dures. Among these 10 articles, three were excluded: for twoarticles we found only the abstract and not the full-text andthe other one was a case report of an 88-year-old multi-diseased osteoporotic patient treated with corticocancellous

bone allograft, augmented with autologous bone marrowconcentrate from iliac crest aspirate enriched with plateletrich fibrin from peripheral blood. Thus, due to the treatmentprotocol and the lack of a control group, the real contribu-tion of MSCs on spinal fusion procedure could not be extra-polated.Therefore, 7 articles were analyzed: by comparing thecharacteristics of each study, it is evident that, in all studies,with the exception of the study by Moro-Barrero et al. [60],the authors employed the concentrate autologous bone mar-row in comparison to fresh one inside the operating theater[20, 61–65]. Three studies associated bone marrow with aceramic graft [20, 60, 61] while the remaining 4 combinedbone marrow with allograft [62–65]. Three studies wereprospective, randomized trials [61–63], two of which withlimited number of patients [61, 62], while one was a prospec-tive, multicenter, nonrandomized study on 182 patients [65].All the studies withdrew the bonemarrow from iliac crest andperformed spinal fusion surgery on 1, 2, or 3 levels with simi-lar surgical procedures and approaches. As far as the numberof transplanted cells, cell concentration was not alwaysreported as cell number in one milliliter or was not reportedat all, thus making comparison among studies extremelydifficult. In addition, another variable among studies is themethod used for cell concentration (cell separator based onthe density gradient centrifugation, centrifugation over a gra-dient, orwithout any gradient).Obviously, the absence of pro-cedural and methodological guidelines affected the cell yieldand thus it was not possible to identify a range for the cellsnumber to be transplanted and to correlate it with the clinicaloutcome. In addition to the cells number, in two studies a con-trol groupwas not used [64, 65], while other two studies com-pared the experimental treatment with autologous bone [60,62] and one study with allograft chips alone [63]. Anotherstudy by Gan et al. compared autologous enriched MSCs/𝛽-TCP with locally harvested bone combined with autologousenriched MSCs/𝛽-TCP [20], while Odri et al. in a simpleblind randomized clinical, prospective, monocentric studycompared a biphasic calcium phosphate ceramics graft thatwas associated with autologous bone and concentrated bonemarrow with unconcentrated bone marrow with ceramicsgraft and autologous bone [61].The follow-up of the analyzedstudies ranged from 12 months to 36.5 months [20, 60–65],demonstrating, through radiographic and clinical analyses,the safety and in one case a greater efficacy [63] of MSCsuse in spinal fusion; however, these studies were conductedin too small patient cohorts and there is the need to con-firm these data also from preclinical animal models, wheretransplanted cell phenotype, fate, and contribution to healingcould be monitored and quantitatively measured to excludemalignant transformation.

As of August 2016, the ongoing clinical trials onMSCs forspinal fusion applications found through https://www.clin-icaltrials.gov web site are 6 (Table 5). One of them wasexcluded because the objective of the trial was the definitionof the osteogenic potential of MSCs and their progenitorsduring spinal fusion complication (pseudarthrosis). Anothertrial has not been analyzed because it has been withdrawnprior to the patients enrollment. The remaining 4 trials were




all interventional study of phase I-II with a minimum follow-up of 12 months. Of them 2 were completed. In detail thetrials assessed (1) the feasibility and safety of ex vivo expandedautologous MSCs fixed in allogenic bone tissue in compar-ison to autologous bone; (2) the effectiveness of autologousmesenchymal stem cells arranged in a calcium phosphateceramic; (3) the effectiveness of allograft alone versus allograftwith bone marrow concentrate; (4) the feasibility, safety, andtolerability of 3 different doses of immunoselected, culture-expanded, nucleated, allogenic mesenchymal precursor cellscombined with resorbable ceramic granules in comparisonto autograft alone. In all studies fusion surgery, surgicalprocedures, clinical approaches, and follow-up evaluationswere similar. However, each trial was different from the otherfor patients number, MSCs manipulation or strategy, studyarms, and presence and/or type of control group. In addition,the information available was not always complete. In somecases it was not clear which strategy would be employed forMSCmanipulation and almost all the studies did not indicatethe number of cells or the medium for cell infusion. Thus,although some studies could provide useful information, itwas evident that more controlled clinical trials are necessaryto understand whether MSCs can be successfully employedin spinal fusion procedures.

5. Conclusion and Future Prospective

In recent years, the basic and preclinical research literatureclearly indicates the use of MSCs also in combination withvarious scaffolds, to repair bone defects, and many stud-ies concern their use also for the treatment of vertebralinstability. Thus, with the rapidly growing number of spinefusion surgeries performed annually, we have seen the needfor performing this descriptive systematic literature reviewon MSCs use in spinal arthrodesis procedures in order toelucidate if the use of MSCs may really represent a validstrategy able to facilitate and accelerate spinal fusion.

In this review, several therapeutic strategies for theenhancement of spinal fusion rate based on stem cells havebeen developed in both preclinical and clinical studies. Theapplication of an allograft or a scaffold, prevalently ceramics,associated with stem cells was adopted in all preclinicalstudies while the application of autograft, but also ceramicscaffolds, still in association with stem cell was used inthe clinical setting (tissue engineering strategy). However,the use of growth factors (principally BMP-2) and otherosteoinductive factors, as well as ex vivo gene therapy, wastaken into consideration.

We found that numerous preliminary researches in thisreview were carried out in small, medium, and large animalmodels showing the potential for MSCs use in spinal fusionprocedures. Despite the fact that in some of these studiesadipose derivedmesenchymal stem cells, human perivascularstem cells, and also amnion epithelial cells were used, themajority of the studies employed bone marrow cells. Basedon these preclinical data it would seem that MSCs are ableto perform the necessary physiological functions to achieve,facilitate, and accelerate spinal fusion.However, none of theseexamined studies was able to give a detailed elucidation about

the fate ofMSCswhen theywere added to a scaffold, althoughthe success demonstrated that, in the animal models, somebarriers remain prior to this therapy translation into the clin-ical setting. In fact, this review underlines that there are fewand basic clinical trials, although some of them have shownthat bone marrow cells used in humans can give a successfulspine fusion. Some critical existing limitations include alsothe choice of the optimal cell concentration, the deliverymethod, the ideal manipulation procedure (ex vivo expan-sion and one-step procedure), and the best implantationtechniques. In addition, researches that examine the optimalMSCs concentration are needed in large animal model,which are more similar to humans. These critical pointsalso highlight the need for methods able to maximize thenumber ofMSCs collected, as well as the presence of easy andfeasible techniques in the clinical scenario. However, othermatters that need further consideration comprise also theelimination of fetal calf serum, the possible reversibility ofthe differentiated state, the survival of the cells in vivo, theintegration with the preexisting bone, and the capacity toform bone and marrow in vivo.

In conclusion, the use of MSCs as a cell-based therapymay represent a biological approach to reduce the high costof osteoinductive factors as well as the high dose needed toinduce bone formation. Thus, implementing this availablepotential treatment based onMSCs use and probablymitigat-ing some adverse effects would make this kind of approacha possible therapeutic tool. Finally, although MSCs ther-apy remains an interesting and important opportunity ofresearch, it is necessary that the spine surgery communitycarefully evaluates the safety and efficacy ofMSCs use in spinefusion through randomized controlled and blinded clinicaltrials.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by grants from Rizzoli OrthopedicInstitute (Ricerca Corrente), 5 × 1000 2013 Project “Sviluppoe Validazione di Modelli Alternativi e Complementari InVitro (Intelligent Testing Strategy) in Ortopedia e Trau-matologia,” Fondazione del Monte di Bologna e Ravenna2016 Project “Cellule Mesenchimali Staminali Autologhe daCorpo Vertebrale come Prospettiva Biologica Innovativa perla Chirurgia Vertebrale,” and the Operational ProgrammeERDF 2007–2013 in the region Emilia-Romagna: Activity 1.1“Creation of Technology Centers for Industrial Research andTechnological Transfer.”

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