Treatment of femoral head osteonecrosis with advanced cell therapy in sheep

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BASIC SCIENCE

Treatment of femoral head osteonecrosis with advanced celltherapy in sheep

Roberto Velez • Alberto Hernandez-Fernandez •

Marta Caminal • Joaquim Vives • Francisco Soldado •

Alejandro Fernandez • Arnau Pla • Marius Aguirre

Received: 10 February 2012! Springer-Verlag 2012

AbstractBackground The purpose of this study was to evaluate the

efficacy of core decompression associated with advanced

cell therapy for the treatment of femoral head osteonecrosisin an established sheep model.

Methods Early stage osteonecrosis of the right hip was

induced cryogenically in 15 mature sheep. At 6 weeks, thesheep were divided into three groups, Group A: core

decompression only; Group B: core decompression fol-

lowed by implantation of an acellular bone matrix scaffold;Group C: core decompression followed by implantation of

a cultured BMSC loaded bone matrix scaffold. At12 weeks, MRI hip studies were performed and then the

proximal femur was harvested for histological analysis.

Results In the group of advanced cell therapy, Group C,there was a tendency to higher values of the relative surface

of newly formed bone with a mean of 20.3 versus 11.27 %

in Group A and 13.04 % in Group B but it was not sta-tistically significant. However, the mean relative volume of

immature osteoid was 8.6 % in Group A, 14.97 in Group B,

and 53.49 % in Group C (p\ 0.05), revealing a greatercapacity of osteoid production in the sheep treated with

BMSCs. MRI findings were not conclusive due to constant

bone edema artifact in all cases.Conclusions Our findings indicate that a BMCSs loaded

bone matrix scaffold is capable of stimulating bone

regeneration more effectively than isolated core decom-pression or in association with an acellular scaffold in a

preclinical femoral head osteonecrosis model in sheep.

Keywords Femoral head osteonecrosis ! Stem cells !Advanced cell therapy ! Bone regeneration

Introduction

Femoral head osteonecrosis, a disease of unknown patho-genesis, is still a clinical challenge to orthopaedic surgeons.

Also many unresolved questions remain for researchers

[1, 2]. Either idiopathic or with an established precipitatingfactor, the disease follows a common route to ischemia and

cell death that can ultimately lead to structural collapse,

articular incongruence and hip osteoarthritis [2–5]. Thereare many hip preserving treatment strategies in the early

precollapse stage like core decompression, osteotomy, and

vascularized or non-vascularized bone grafting, but the

R. Velez (&) ! F. Soldado ! A. Fernandez ! M. AguirreOrthopaedic Surgery and Traumatology,Hospital Universitario Vall dHebron,Pg Vall d’Hebron 129-139, 08035 Barcelona, Spaine-mail: [email protected]

F. Soldadoe-mail: [email protected]

A. Fernandeze-mail: [email protected]

M. Aguirree-mail: [email protected]

A. Hernandez-FernandezOrthopaedic Surgery and Traumatology, Hospital Donostia,San Sebastian, Spaine-mail: [email protected]

M. Caminal ! J. Vives ! A. PlaBanc de Sang i Teixits, Divisio de TerapiesAvancades/XCELIA, Barcelona, Spaine-mail: [email protected]

J. Vivese-mail: [email protected]

A. Plae-mail: [email protected]

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Arch Orthop Trauma Surg

DOI 10.1007/s00402-012-1584-6

Author's personal copy

results are still not optimal [5–8]. The only treatment

strategy currently available for late stage osteonecrosiswith femoral head collapse is resurfacing or total hip

arthroplasty [9–11]. It is therefore imperative to focus

research on the development of bone regenerative strate-gies in the early precollapse stages of the disease that can

progressively decrease the need for total hip replacement

for this young, productive patient population [1]. Someclinical studies have already used bone marrow derived

mesenchymal stem cells (BMSCs) as an osteogenicinduction factor [12–15]. Considering the inherent patho-

genic factors of the disease including deficient quantitative

and qualitative ratios of BMSCs in these patients, it hasbeen proposed that in vitro amplification of BMSCs could

improve these results [16].

We recently developed a femoral head osteonecrosisanimal model in sheep which replicated all the stages of the

disease and that has been considered useful for preclinical

research [17, 18]. Current trends in the management ofearly stage osteonecrosis focus on core decompression

associated with biologic adjuncts including an advanced

cell therapy modality with cultured BMSCs [1, 4, 5, 19, 20].Our hypothesis was that a BMSC culture loaded bone graft

could stimulate bone regeneration in an established osteo-

necrosis femoral head sheep model.

Materials and methods

All experimental procedures adhered to the recommenda-

tions of local, national, and European laws (Decret 214 of1997, Real Decreto 223 of 1988, European directive

86/609/CEE of 1986, respectively) and to the standards of

care set by the National Institutes of Health (Guide for Careand Use for Laboratory Animals, publication No. 80-23,

revised 1985). Our project also received the approval of the

Ethics Committee of the Institut de Recerca del HospitalUniversitario Vall d0Hebron.

Establishment of the femoral head osteonecrosis

We obtained 15 mature sheep from a licensed provider of

experimental animals. Osteonecrosis was induced in theright hip according to our published animal model [17].

All sheep were anesthetized and placed in decubitus supine

position. The medial aspect of the hip was prepared and thehip joint was exposed through a 15 cm incision over the

inguinal sulcus. After vascular ligation, the hip capsule was

incised and the inferior femoral neck exposed. Then atunnel was opened in a superior direction using a 4.5 mm

drill bit and a 2 mm cryoprobe (Mini-probe; Brymill,

Ellington, CT, USA) was introduced and attached to aliquid nitrogen pistol (CRY-AC; Brymill, Ellington, CT,

USA). Correct placement of the cryoprobe tip in the

anterosuperior area of the femoral head was checked withan intraoperative image intensifier. The cryoprobe tip was

cooled for 9 min followed by surgical field irrigation with

warm saline (50 "C). This freeze–thaw cycle was repeatedthree times. The cryoprobe was then removed and the

tunnel was sealed with bone wax. The surgical approach

was then closed in anatomical layers.

Culture of BMSCs and colonization of the scaffold

Three weeks after the osteonecrosis induction and with the

same anesthesia protocol, the sheep were placed in decu-bitus position and the sternum was aseptically prepared and

draped. Using an 11G trochar, 2 mL of bone marrow was

aspirated before redirectioning the trochar and re-aspirat-ing. A total of 65 cc of bone marrow aspirate was obtained

and collected in a flask with 8 mL of ACD-A. The bone

marrow was then diluted with PBS solution and thencentrifuged in Ficoll for 40 min to isolate the mononuclear

cell band. These cells were then cultured in Dulbeccos

modified eagle medium and supplemented with autologousserum. Cells were cultured for 21 days achieving an

average 6 million cells/cc. The cells were then left to

colonize a malleable cylinder composed of 2 g of lyophi-lized trabecular bone graft (Osteoplant; Bioteck, Vicenza,

Italy).

Core decompression and treatment groups

Six weeks after the induction of the femoral head osteo-necrosis, the 15 sheep were again anesthetized under the

same protocol and placed in a decubitus supine position.

The lateral aspect of the hip was then aseptically preparedand draped. A small percutaneous incision was made just

distal to the greater trochanter accessing the lateral femoral

cortex. Using an image intensifier, a 2-mm guide wire wasadvanced from the lateral cortex to the anterosuperior

region of the femoral head reaching the periphery of the

necrotic lesion. A 5-mm drill bit was then advanced overthe guide wire again to the periphery of the lesion. The drill

bit and the guide wire were then removed. A 5 mm tre-

phine was then introduced through the tunnel and advancedthrough the necrotic lesion. The trephine was removed and

the bone cylinder extracted was placed in formaldehyde

and reserved for histological analysis. In the core decom-pression group of 5 sheep, Group A, no additional proce-

dures were done and the surgical approach was closed in

standard fashion. In the biological treatment groups afterthe core decompression, the surgical procedure continued

as follows. In the scaffold only group of 5 sheep, Group B,

the scaffold was prepared and introduced in the followingmanner. Two grams of lyophilized trabecular bone graft


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(Osteoplant; Bioteck, Vicenza, Italy) was introduced

within a sterile 5 mm trephine and thrombin and fibrinogen(Tissucol Duo; Baxter, Deerfield, IL) were added to

solidify the construct. Then trephine was introduced

through the tunnel reaching the periphery of the necroticlesion and an obturator was pushed through the trephine

expelling the bone graft within the necrotic lesion. The

empty trephine was then removed and the surgicalapproach closed. In Group C, the BMSC group, 5 sheep

were also used. Using the same technique as in the scaffoldonly group, the BMSC culture loaded bone graft was

combined with thrombin and fibrinogen (Tissucol Duo;

Baxter, Deerfield, IL) within a 5 mm trephine which wasthen applied to the necrotic lesion in similar fashion.

Magnetic resonance imaging studies

MR imaging followed our previously published protocol

using a 0.2-T system (VET-MR; Esaote, Genoa, Italy) [17].We examined the hip under general anesthesia using the

same anesthesia protocol as for the osteonecrosis inducing

procedure. MR imaging was obtained 6 weeks after theinduction of osteonecrosis, just before the surgical treat-

ment, and later at 12 weeks (6 weeks after surgical treat-

ment). MR imaging of the contralateral hip was obtained intwo sheep as control. Femoral head osteonecrosis was

staged according to the Ficat and Arlet and the Association

Research Circulation Osseous (ARCO) classification sys-tems. [21].

Histological analysis

Six weeks after the three allocated treatment groups, all 15

sheep were killed. Histological analysis was performed onboth the cylindrical bone samples recollected from all the

core decompressions at the beginning of the different treat-

ments and on all proximal femurs harvested at the end of thestudy. The specimens were fixed in 10 % buffered neutral

formalin and decalcified. The specimens were then embed-

ded in paraffin. Coronal sections in 4 lwidth were cut usingan electronic microtome and stained with haematoxylin and

eosin. The extent of early osteonecrosis was evaluated in the

cylindrical bone samples taking into account empty lacunaeand fibrosis. The femoral heads at the end of the treatment

modalities were evaluated in the superior section measuring

bone regeneration around the proximal end of the tunnel(core decompression only group) or the proximal tip of the

scaffold (scaffold only and scaffold plus BMSCs groups).

These sections were then digitalized and analyzed usingImageJ software (National Institutes of Health, US Gov-

ernment) and the plug-in BoneJ. The relative surface of

newly formed bone of the total area of tissue was calculated,using a 8-bit image input with the command Optimise

Threshold. The total bone area (BA) was divided by the total

area of tissue (TA), after the area of lamelar bone (LBA) wassubstracted by thresholding from the total bone surface (BA)

to obtain the non-lamelar relative surface (NB/TA), thus, to

estimate the rate of newly formed bone. The relative volumeof immature osteoid was also measured using a 100 point

reticle in which all points presenting immature osteoid were

added and expressed as a percentage [22].Statistical analysis with non-parametric tests was per-

formed using SPSS 18.0 (IBM, Armonk, USA) statisticssoftware.

Results

In the initial 48 h after all the hip surgical procedures,including the osteonecrosis induction and the treatment

modalities, the sheep showed mild protected weight bear-

ing. Afterwards all of the fifteen sheep walked normallywithout evidence of limp. Three sheep developed a local

presternal hematoma after the bone marrow aspiration

procedure that resolved without further treatment. Nomajor complications like fractures or surgical site infec-

tions were evidenced during the study period.

MR imaging

The control studies showed normal findings in the contra-lateral hip. Six weeks after the osteonecrosis induction,

MRI showed typical early stage osteonecrotic changes in

all the hips with no femoral head collapse or joint spacenarrowing. All lesions were classified as stage 1 of both the

Ficat and Arlet and the ARCO classifications. At 12 weeks,

6 weeks after the different treatments, MRI showed nodifferences between the groups. There was a homogeneous

bone edema of the proximal femur secondary to the core

decompression (Fig. 1). None of the hips progressed tocollapse, articular incongruence, or joint space narrowing

at this time.

Histological assessment

In all the 15 bone cylinders extracted during core decom-pression six weeks after the femoral head osteonecrosis

induction technique, osteocytes were absent within the

lacunae and the normal marrow had been replaced by pro-gressive fibrosis consistent with early stage femoral head

osteonecrosis. The macroscopic analysis of the 15 proximal

femurs showed no femoral head collapse or articularincongruence. All the samples obtained from the 5 sheep

treated with core decompression alone, and the 5 sheep

treated with core decompression associated with the bonegraft implant showed persistence of osteonecrotic changes,


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including empty lacunae and extensive fibrosis. There wasminimal presence of immature osteoid, new bone, or cellular

regeneration in all the samples (Fig. 2). Samples from two

sheep treated with core decompression and bone graftingshowed some zones of foreign body reaction as seen with

some evidence of granuloma formation. In the group treated

with core decompression and implantation of BMSC loadedbone graft, 80 % of the samples showed extensive bone

regeneration. There was evidence of osteoid infiltration of

the necrotic and ischemic areas (Fig. 2f). The maturelamellar bone in these constructs was surrounded by large

numbers of osteoblasts producing immature osteoid (Fig. 3).

There was also less evidence of fibrosis and no signs ofgranuloma formation in these samples.

The image software analysis of the histologic samples

revealed that the group of advanced cell therapy, Group C,has a tendency toward higher values of the relative surface

of newly formed bone with a mean of 20.3 % (14.01–27.20 %)

versus 11.27 % (7.88–17.81 %) in Group A and 13.04 %(6.90–21.54 %) in Group B but it was not statistically

significant. Osteoid production as measured by the relative

volume of osteoid was dramatically increased in theBMSCs group. The mean relative volume of immature

osteoid was 8.6 % (5.21–11.20 %) in Group A, 14.97 %

(4.82–32.74 %) in Group B, and 53.49 % (47.45–59.32 %)in Group C (p\ 0.05)(Fig. 4).

Discussion

Following current trends in the treatment of osteonecrosisand the fundamental guidelines in tissue engineering

techniques for bone regeneration, our research group set

out to identify the optimal components of a biologicalconstruct as an addition to the standard care with core

decompression. Many authors propose that modern bio-

logic techniques associated with core decompression forsmall and moderate size necrotic lesions should be the

focus of new regenerative therapies [4, 5, 19, 20, 23, 24].

Adding a biologic construct to a percutaneous coredecompression technique can be easily transferred to the

clinical setting, considering that this technique has lowmorbidity and is minimally invasive [20, 23]. Considering

what is proposed as the optimal biological treatment

in bone regeneration that includes the basic pillars ofosteoproduction, osteoconduction, and osteoinduction, we

designed a bone graft scaffold loaded with cultured BMSCs

to evaluate its efficiency against simple core decompres-sion or its association with an acellular bone graft [25].

Reports show that augmenting osteoproduction through

concentrated BMSCs seems to increase the effectiveness ofcore decompression, but also that this technique has its

limitations since many patients with osteonecrosis have

poor mesenchymal cell concentrations and that these cellsalso have decreased differentiation capacity [12–14,

26–28]. To overcome these obstacles, we have increased

the number of BMSCs through ex vivo expansion instandard fashion, a recommendation already proposed in

the literature [16]. Ex vivo expansion allows for a cell

quality control that preestablishes the adequate number ofstem cells, with differentiating capacity, that colonize the

bone graft. The group of sheep treated with the BMSC

loaded bone graft was the only group that stimulated boneregeneration within the ischemic area of the femoral head.

The addition of cultured stem cells really set the difference

in osteoid production locally in an hypoxic environment.This higher osteoid concentration translated into an

apparent higher tendency to definitive new bone formation.

We believe that the tendency to higher new bone formationwould have been statistically significant if the treatment

had been prolonged longer than the established 6 weeks.

These results show that the addition of cultured andexpanded BMSCs ex vivo through advanced cell therapy

techniques to a standard core decompression of femoral

head osteonecrosis stimulates immature bone formation denovo much more effectively than the other two techniques.

There are preclinical reports on the use of BMSCs in the

treatment of femoral head osteonecrosis with good results,but most of these have been performed on animal models

that do not reproduce all stages of human disease [18,

29–31]. BMSCs have also been found to survive, prolif-erate, and differentiate into osteoblasts directly in an

ischemic femoral head and seem to contribute to an

acceleration in the repair process [32]. Recently, Tangreported on the treatment of osteonecrosis of the femoral

Fig. 1 T2 weighted MRI of the right hip 6 weeks after core-decompression and implantation of advanced cell therapy treatment,Group C showing extensive edema in the proximal femur


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head with gene-modified tissue-engineered bone in an

established animal model in goats, in which BMP-2-gene-

transduced BMSCs were shown to be capable of repairingearly stage, experimentally induced osteonecrosis. The

authors suggested that the osteoinduction through BMP-2

gene therapy, which augmented local concentrations, could

effectively restore the mechanical function of the repara-

tive tissue to prevent the femoral head from collapsing in

patients with osteonecrosis [33]. It must be stated thatalthough genetically engineered bone tissue could lead to

future developments in therapies for osteonecrosis, its

immediate translational application could be difficult

Fig. 2 Samples obtained 6 weeks after treatment allocation. Histo-logical specimen after core decompression (Group A) with fibroticmarrow and empty lacunae, consistent with advanced osteonecroticchanges (a, b). Histological sample after core decompression andtreatment with a bone graft scaffold (Group B). The mature lamellarbone graft has not stimulated osteoid production and the osteonecrotic

changes are maintained. (c, d) Histological sample after coredecompression and treatment with an advanced cell therapy loadedscaffold (Group C) showing an ischemic area (top half) that showsprogressive bone regeneration through and immature osteoid front.(e, f). Hematoxylin–eosin, 925 (a, c, e) Hematoxylin–eosin, 975(b, d, f)


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considering cells transduced with adenovirus express viralproteins on their cell surface, which could trigger cell-

mediated immunity [34]. As our results show bone regen-

eration can be obtained without local augmentation of os-teoinductors through gene modifications. Our bone graft

scaffolds contain small concentrations of osteoinductors

that seem to provide sufficient beneficial local stimuli [35].Scaffold selection is also important for its osteoconduc-

tive properties and structural support. Besides bone grafting

techniques, different types of scaffolds have also been usedpreclinically and clinically in femoral head osteonecrosis,

including b-tricalcium phosphate, allograft threaded cages,

hydroxyapatite, and porous tantalum implants [5, 33, 36,37]. Our scaffold, a freeze-dried cancellous bone matrix,

allowed us to obtain a malleable cylinder combined with

fibrin which adapted to the dimensions of the tunnel aftercore decompression. Allogenic cancellous bone grafts are an

osteoconductive solution commonly used orthopaedic sur-

gery, and in contrast to structural cortical grafts whichweaken in the early incorporation, the cancellous chips tend

to start strengthening the construct from the start [35]. Fibrin

is supported as a versatile biopolymer with great potential incombination with other elements for tissue regeneration,

that apart from improving the mechanical properties of theconstruct, it stimulates angiogenesis, cell adhesion and

proliferation [38]. We think that apart from these biological

advantages, a failed cancellous graft-based scaffold shouldnot affect a future total hip replacement, or present concerns

of remnant debris, as would be case of vascularized fibular

grafts or tantalum implants [5, 39]. Since the samples col-lected from the isolated scaffold group did not stimulate

osteoid production locally or induce bone maturation we

believe that its osteoconductive and osteoinductive proper-ties when used without cultured BMSCs are insufficient for

bone regeneration. It is when the scaffold is loaded with

BMSCs that all three factors of bone regeneration acttogether to produce good results.

The development by our research group of a predictable

osteonecrosis animal model enabled us to generate threehomogeneous groups with early stage osteonecrosis, stage 1

Ficat and Arlet and ARCO, that would have enabled us to

assess the therapeutic effects with MRI. [17, 21] Unfortu-nately, our study was unable to evaluate through MRI the

therapeutic effects of the different treatment modalities.

In our opinion, this was due to the limited time between thesurgical treatment and the follow-up MRI, where there was

an important artifact of edema that prevented the proper

display of the areas with viable bone or necrosis, moreover,

Fig. 3 Histological sample showing mature lamellar bone graftsurrounded by osteoblasts producing profuse immature osteoid in asheep treated with a cultured stem cell loaded implant. Hematoxylin–eosin, 9100

Fig. 4 Left box plot showing a higher tendency of newly formed bone in Group C. Right box plot showing the augmentation of mean relativevolume of immature osteoid through BMSC’s


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the low power of the apparatus used (0.2 Tesla). Other

authors have used devices 7–8 times more powerful, allow-ing a detailed study of the lesion [40]. Since MRI is the gold

standard in diagnosis and follow-up of femoral head osteo-

necrosis, refinement of a suitable time to evaluate themechanical impact of bone regeneration and the use of more

powerful imaging devices should be the future step [3, 5].

From our histological results, it seems that only atreatment that combines the structural and inductive

properties of bone graft together with the bone productivecapacity of cultured and expanded BMSCs can effectively

regenerate bone in a femoral head osteonecrosis model in

sheep. This treatment option should be the focus of newclinical trials to objectively analyze its capacity to alter the

natural course of the disease.

Acknowledgments This project received financial support from theMinisterio de Educacion, Gobierno de Espana (Spanish Minsistry ofEducation, Madrid, Spain) and from Fundacio Ferrer Investigacio andFundacio Privada A. Bosch (Beguda, Spain). The authors thank MartaRosal, Marielle Esteves, Alex Rojo and all the personnel at the Institutde Recerca-Hospital Unversitari Vall d’Hebron involved in theproject.

Conflict of interest The authors declare they have no conflict ofinterest.

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Treatment of femoral head osteonecrosis with advanced cell therapy in sheep

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