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ResearchCite this article: Jeon JE, Vaquette C,

Theodoropoulos C, Klein TJ, Hutmacher DW.

2014 Multiphasic construct studied in an

ectopic osteochondral defect model. J. R. Soc.

Interface 11: 20140184.

http://dx.doi.org/10.1098/rsif.2014.0184

Received: 21 February 2014

Accepted: 10 March 2014

Subject Areas:biomaterials, biomedical engineering

Keywords:multiphasic scaffold, biomaterials,

osteochondral repair, tissue engineering,

in vivo model

Author for correspondence:Dietmar W. Hutmacher

e-mail: dietmar.hutmacher@qut.edu.au

& 2014 The Author(s) Published by the Royal Society. All rights reserved.

Multiphasic construct studied in anectopic osteochondral defect model

June E. Jeon, Cedryck Vaquette, Christina Theodoropoulos, Travis J. Kleinand Dietmar W. Hutmacher

Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue,Kelvin Grove, Queensland 4059, Australia

In vivo osteochondral defect models predominantly consist of small animals,

such as rabbits. Although they have an advantage of low cost and manageabil-

ity, their joints are smaller and more easily healed compared with larger

animals or humans. We hypothesized that osteochondral cores from large

animals can be implanted subcutaneously in rats to create an ectopic

osteochondral defect model for routine and high-throughput screening of mul-

tiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine

osteochondral plugs with 4 mm diameter osteochondral defect were fitted

with novel multiphasic osteochondral grafts composed of chondrocyte-

seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior

to being implanted in rats subcutaneously with bone morphogenic protein-7.

After 12 weeks of in vivo implantation, histological and micro-computed tom-

ography analyses demonstrated that TECs are susceptible to mineralization.

Additionally, there was limited bone formation in the scaffold. These results

suggest that the current model requires optimization to facilitate robust bone

regeneration and vascular infiltration into the defect site. Taken together, this

study provides a proof-of-concept for a high-throughput osteochondral

defect model. With further optimization, the presented hybrid in vivo model

may address the growing need for a cost-effective way to screen osteochondral

repair strategies before moving to large animal preclinical trials.

1. IntroductionArticular cartilage is a unique tissue, which lacks vasculature, and has limited

self-healing ability. When left untreated, cartilage damage caused by trauma or

other degenerative conditions will not only cause pathological changes to the

surrounding cartilage, but also to the subchondral bone [1]. Osteochondral

lesions, which allow bone marrow infiltration into the damaged site, have

better regenerative capabilities but they often result in fibrocartilage with

inferior mechanical properties and diminished long-term durability. Current

treatment strategies for osteochondral lesions, such as joint debridement and

microfracture, have the disadvantage of fibrocartilage formation [2]. Osteochon-

dral autografts or mosaicplasty are also commonly used to treat osteochondral

defects and often have better outcomes, but these procedures have problems of

donor tissue availability, topology mismatch and donor site morbidity [3].

Autologous chondrocyte implantation (ACI) and matrix-associated ACI

(M-ACI) are some of the cell-based treatment methods that have shown positive

outcomes [4,5] but they also fall short of fully recapitulating the characteristics

of the native tissue [6].

In order to further improve current cell-based treatment methods, such as

ACI and M-ACI, the effects of combining other factors such as scaffolds

(cellular solids), matrices (hydrogels), growth factors and mechanical stimuli

are actively being investigated [7]. When combined with growth factors such

as TGF-b1, hydrogels such as alginate [8] and agarose [9] have been shown

to help chondrocytes maintain their spherical morphology while supporting

chondrogenic differentiation and cartilage-specific matrix deposition. Cartilage

constructs can also be mechanically stimulated in vitro to enhance chondrocyte

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matrix synthesis and remodelling [10,11], and to recapitulate

zonal characteristics in the construct [12,13]. For bone tissue

engineering, polymeric and ceramic biomaterials are com-

monly used to design and fabricate scaffolds [14] with the

addition of osteogenic growth factors, such as bone morpho-

genic protein (rh-BMP)-2 or rh-BMP-7 and bone-forming cells

(e.g. osteoblasts) [15]. One of the most recent developments

in osteochondral tissue engineering is the emergence of not

only biphasic but multiphasic scaffolds [16]. By integrating

cartilage- and bone-specific matrices and scaffolds, which

are tailor-made to enhance chondrogenic or osteogenic devel-

opment of the construct, multiphasic tissue-engineering

approaches aim to enhance the integration and regenerative

potential of the engineered grafts.

With an increasing number of concepts emerging in

the cartilage and osteochondral tissue-engineering disciplines,

there is a growing need for high-throughput and cost-

effective in vivo animal models. Rabbits are commonly used

for osteochondral defect studies, but their joint scales are

much smaller than humans [17] (cartilage thickness: approx.

0.3 mm [18]), and osteochondral lesions in smaller animals,

such as rabbits, are known to have the tendency to heal

more readily compared with larger animals [19]. As a result,

treatment strategies developed using small animals may not

be as effective in larger animals, and there are obvious limit-

ations in interpreting and extrapolating treatment outcomes

from small-animal studies [20]. To this end, it is important

to scale up and use larger animal models with comparable

cartilage thickness and joint scale as humans for osteochon-

dral tissue-engineering studies [21]. However, large animal

models (e.g. porcine, dog or equine) are relatively expensive

and facilities that can accommodate them may not be readily

accessible, which make them less feasible in routine screening

of newly developed tissue-engineering strategies. To bridge

the gap of cost effectiveness by maintaining larger sample

size, accessibility and anatomical tissue scales, we hypo-

thesized that osteochondral cores from large animals, with

osteochondral defects, implanted subcutaneously in rats

would allow for screening of osteochondral repair potential

of multiphasic constructs.

2. Material and methods2.1. Chondrocyte and osteoblast isolation and cultureChondrocytes were isolated from macroscopically normal parts

of the cartilage from patients undergoing joint replacement surgeries

with ethical approval from Queensland University of Technology

and Prince Charles Hospital (n ¼ 3). Superficial (S) and middle-

deep (MD) zone chondrocytes were isolated, as described previously

[22]. This technique has been optimized in our laboratory and

involves manual dissection of the two layers using scalpel blades.

Diced S and MD cartilage pieces were digested overnight in 0.15%

collagenase type 2 (Worthington, Lakewood, NJ, USA) in serum-

free basal medium (LG-DMEM, 2 mM GlutaMax, 110 mg l21

sodium pyruvate, 50 U ml21 penicillin, 50 mg ml21 streptomycin).

All reagents were obtained from Invitrogen (CA, USA) unless

noted otherwise. Isolated chondrocytes were filtered through cell-

strainers (100 mm), washed using phosphate-buffered saline (PBS)

and seeded in T175 flasks at a density of 3000 cells cm22 for expan-

sion up to 1 passage (P1). Primary osteoblasts were isolated from the

bone chips collected from the macroscopically normal bone off-cuts

obtained from the joint replacement surgery patients. Bone chips

were washed 10 times by vigorous shaking in PBS and incubated

in 0.25% trypsin-EDTA for 30 min. After another wash in PBS,

bone chips were cultured in T175 flasks. Chondrocytes and osteo-

blasts from each patient were cultured separately using the basal

medium supplemented with 10% fetal bovine serum (FBS, Hyclone,

UT, USA), and media (15–20 ml) were changed twice a week. Once

confluent, cells were passaged using 0.1% trypsin-EDTA.

2.2. Chondrogenic differentiation of alginate discsseeded with surface or middle-deep chondrocytesand mechanical stimulation (cartilagecompartment)

After expansion, S and MD chondrocytes (P1) were resuspended

in 2% w/v alginate (Pronova UP LVG, FMC biopolymers, PA,

USA) solution at 107 cells ml21 and cross-linked using a

custom-made mould immersed in 102 mM CaCl2 bath [22].

Each S and MD construct was 4 mm and 1.5 mm in diameter

and thickness, respectively. Alginate discs were placed in

24-well plates and cultured in serum-free chondrogenic media

(HG-DMEM (Invitrogen), 1.25 mg ml21 bovine serum albumin,

1% ITS þ 1, 1027 M dexamethasone, 0.1 mM L-ascorbic acid (all

Sigma, USA) and 10 ng ml21 TGF-b3 (GroPrep Bioreagents, Ade-

laide, Australia)) for 12 weeks with media changed twice per

week (0.6 ml/construct). Following the 12-week culture, S and

MD alginate constructs were divided into either non-

compressed (NC) or compressed (Comp) groups. Constructs

selected for loading were subjected to a one-week intermittent

dynamic compression in a commercial unconfined compression

bioreactor (Cartigen C10–12c, Tissue Growth Technologies, Min-

netonka, MN, USA) while those in the NC group were cultured

under free-swelling conditions. The loading protocol was chosen

based on a previous optimization study for chondrocytes in algi-

nate gels [12] and consisted of 1 Hz sinusoidal compression for

3 h d21 at 50% compressive strain.

2.3. Biphasic construct fabrication, assembly andosteoblast seeding (bone compartment)

For the first layer, medical-grade polycaprolactone (PCL) was used

to fabricate a composite scaffold via fused deposition modelling

(FDM, Osteopore Inc., Singapore). The scaffolds measured 100 �100 � 6 mm3 and had 100% interconnectivity, 70% porosity and

a 08/908 lay-down pattern. For the second layer, medical-grade

PCL pellets (Lactel, USA) were loaded into a 2 ml syringe and

were electrospun using an in-house melt electrospinning device

(4 cm tip-to-collector distance, 808C, 20 ml h21, 7 kV) [23,24].

After 4 min of electrospinning, highly porous PCL melt-electro-

spun mesh with roughly 8 mm diameter was produced on top

of the aluminium foil-covered glass slides placed over the collector.

The biphasic scaffold was assembled as previously reported

[25,26]. Briefly, 6 mm long FDM scaffold cylinders were punched

out using a 4 mm biopsy punch and one side of the cylinder

was placed 1 cm from a hot plate heated to 3008C for 4 s and

then quickly press-fitted for 10 s onto the pre-cut PCL meshes

(4 mm diameter, approx. 1 mm thick). This heat treatment partially

melted the first layer of the FDM component enabling it to strongly

bind to the electrospun scaffold upon cooling and solidification.

Once the biphasic scaffolds were fused, they were soaked in 1 M

NaOH solution overnight to increase hydrophilicity. Biphasic scaf-

folds were then washed in 70% ethanol and put under UV for

20 min to sterilize and were left to dry in a sterile laminar hood

overnight. Once dry, biphasic scaffolds were placed in 24-well

plates and each scaffold was seeded with 2.5 � 105 osteoblasts

in 100 ml of basal media supplemented with 10% FBS. Cells

were allowed to adhere to the scaffold for 4 h in the incubator

before adding additional media (1.5 ml/construct). Scaffolds

electrospun

PCL scaffold

bovi

ne b

one

bovi

ne b

one

bovi

neca

rtila

ge

bovi

neca

rtila

ge

PCL-FDMscaffold

2%alginate

BMP-7

electrospun PCL meshS

MD2 mm

6 mm

1 mm

4 mm

(b)

(a)

(c) (d )

Figure 1. (a) Overview of the experimental design. Triphasic constructs were composed of chondrocyte-seeded bilayered (S, MD) 2% alginate gels and osteoblast-seeded PCL biphasic scaffolds. Alginate gels were cultured in chondrogenic media for 12 weeks and subjected to one week of compressive stimulation prior to in vivoimplantation. Triphasic constructs seeded with chondrocytes and osteoblasts were imaged using stereo microscopy, confocal microscopy (rhodamine – phalloidin(red), DAPI (blue)) and scanning electron microscopy (b) (scale bar, 1 mm). Prior to the rat subcutaneous implantation, osteochondral cores prepared frombovine knees were filled with triphasic constructs and the cartilaginous compartment was covered with electrospun PCL mesh (c) (scale bar, 6 mm). Osteochondralconstructs were implanted in rat subcutaneously in the dorsal pouches underneath the skin (d ) and allowed to mature for 12 weeks.

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were cultured for 24 h prior to the final assembly into triphasic

osteochondral constructs.

2.4. Preparation of bovine osteochondral defects andtriphasic construct assembly

An adult bovine osteochondral joint was obtained from a local

abattoir. The joint was securely clamped on top of a surgical

bench and the joint capsule was opened using sterile scalpel

blade and surgical forceps. Osteochondral plugs of 6 mm in diam-

eter were cut from the patellofemoral groove using a sterile

trephine and a hacksaw. The tissue was constantly irrigated

with sterile PBS containing antibiotics to minimize heat-induced

damage. To create 4 mm diameter full thickness defects, osteo-

chondral plugs (n ¼ 32) were clamped down and were drilled in

the centre using a sterile trephine. The defect plugs were then cul-

tured in basal media containing 10% FBS for 3 days until the

triphasic construct insertion and rat subcutaneous implantation.

The triphasic construct was assembled 24 h prior to the

in vivo implantation using a modified protocol from a previous

study [22]. First, S and MD alginate discs (Comp or NC) were

placed on sterile filter paper soaked with 200 mM EDTA for 3 s

to partially de-cross-link one side on each disc. The de-cross-

linked sides were then pressed together for 10 s before being

transferred to a 102 mM CaCl2 bath for 10 min. To combine the

alginate constructs with the PCL biphasic scaffolds, the MD

side of the combined alginate was partially de-cross-linked

using 200 mM EDTA-soaked filter paper and was press-fitted

on top of the FDM side of the biphasic scaffold for 10 s to

allow alginate to partially infiltrate the pores of the PCL-FDM

scaffolds and were re-cross-linked in 102 mM CaCl2 bath. Com-

bined triphasic constructs were cultured in basal media

supplemented with 10% FBS for 24 h prior to combining with

the osteochondral defect plugs and being implanted in rat

subcutaneously. Cell-free alginates and biphasic PCL scaffolds

were also prepared using the same method, as controls.

2.5. Subcutaneous implantation of multiphasicconstructs in rat

Figure 1a depicts the global strategy conducted in this study,

which consisted of inserting a triphasic construct (figure 1b)

in a bovine osteochondral defect plug (figure 1c) prior to subcu-

taneous implantation in a rodent model (figure 1d ). Immediately

before the surgical implantation, triphasic constructs were taken

out of the media, gently dried using sterile gauze and inserted

into the bovine osteochondral defect plugs. 15 ml of recombinant

human bone morphogenic protein-7 (rhBMP-7, 1 mg ml21,

Olympus Biotech Corporation) mixed with 15 ml of fibrinogen

and 15 ml of thrombin (TISSEL, Baxter, IL, USA) was injected

into the bone compartment of the osteochondral defect. A thin

PCL electrospun membrane (less than 0.5 mm thick, 8 mm diam-

eter) was glued on top of the cartilage/alginate side using 5 ml of

fibrinogen and 5 ml of thrombin to give an added protection

against fibrous tissue infiltration into the cartilage compartment.

Bovine defect plugs containing the triphasic constructs and

rhBMP-7 were then implanted subcutaneously in athymic nude

rats. This experimental design did not consider the implantation

of osteoblast-seeded only (no rh-BMP-7) triphasic scaffolds as no

ectopic bone formation was expected in this group, as we have

recently demonstrated [27]. Animal ethics approval for the use

of rats in this experiment was granted by the Animal Ethics

Committee of Griffith University. Four 8-week-old male rats

(Animal Resources Centre, Canning Vale, WA, Australia) were

anaesthetized with isoflurane, and eight incisions were made

longitudinally along the central line of the shaved dorsal area,

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approximately 1.5 cm apart, and subcutaneous pockets were

made with a pair of surgical scissors. The assembled bovine

defect plugs were inserted individually in each subcutaneous

pocket. Rats were allowed to move freely within the cage and

were closely monitored throughout the experiment. Following

12 weeks of implantation, animals of all groups were sacrificed,

and the bovine defect plugs were fixed in 4% paraformaldehyde

(PFA) for 2 h. Fixed explants were incubated in a solution of

50 mM BaCl2 and 100 mM sodium cacodylate trihydrate over-

night to stabilize the remaining alginate gels and kept in 0.1%

sodium azide in PBS solution at 48C prior to further analysis.

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2.6. Analysis of glycosaminoglycan, DNA andmechanical properties of superficial andmiddle-deep alginate constructs

After 12 weeks of chondrogenic culture and one week of loading,

S and MD alginate constructs (NC and Comp) were analysed for

glycosaminoglycan (GAG) and DNA content (n ¼ 6), and also

compressive modulus (n ¼ 6). Prior to the biochemical assays,

S and MD constructs were digested in 0.5 mg ml21 proteinase

K solution (Invitrogen) overnight at 608C and thoroughly

mixed using the vortex. A modified 1,9-dimethylmethylene

blue (Sigma) assay at pH 1.5 [28] was used to determine GAG

content. DNA content in the S and MD constructs was quantified

using the Quant-iT PicoGreen dsDNA assay kit (Invitrogen)

according to the manufacturer’s protocol. The absorbance read-

ing from the GAG plate and the fluorescence reading from the

PicoGreen kit were measured on a POLARstar microplate

reader (BMG Labtech, Mornington, Australia). The compressive

moduli of the constructs were measured on an Instron 5848

microtester fitted with a 5 N load cell (Instron, Australia).

S and MD constructs were kept in media until immediately

before the compression test. The compression testing protocol

comprised a slow downward ramp (10 mm s21) from the contact

point (0.02 N) until 50% compressive strain level was reached.

The slope of the stress–strain curve in the 10–15% strain range

was used to calculate the compressive modulus.

2.7. Immunofluorescence staining of superficial andmiddle-deep constructs

In order to visualize the accumulation of collagen types I and II

following the long-term chondrogenic culture and loading, S and

MD alginate gels were immersed in a fixative solution containing

4% w/v PFA, 100 mM sodium cacodylate trihydrate (Sigma) and

10 mM CaCl2. To stabilize the alginate during the processing

steps, constructs were incubated at 48C overnight in 50 mM

BaCl2 solution containing 100 mM sodium cacodylate trihydrate.

Fixed constructs were paraffin-embedded and sectioned at 5 mm.

Once de-paraffinized and rehydrated, sections were soaked in

10 mM sodium citrate/0.05% Tween 20 buffer at pH 6.0 and

heated in a pressure cooker for 4 min at 948C. Heat-treated sec-

tions were further incubated with 0.1% w/v pronase and 0.1%

w/v hyaluronidase (all Sigma) solution at 378C for 30 min.

After the antigen retrieval process, sections were blocked with

2% FBS solution for 1 h and incubated overnight in primary

antibody solution for collagen type I (Col I, dilution 1 : 500,

I-8H5, MP Biomedicals, USA) and collagen type II (Col II,

dilution 1 : 200, II-II6B3, Developmental Studies Hybridoma Bank

(DSHB), USA). Sections were washed twice in PBS and incubated

in fluorescence-labelled goat anti-mouse secondary antibody

(Alexa Fluor 488, Invitrogen) for 1 h prior to mounting with Prolong

Gold (Invitrogen). After allowing the mounting media to dry in the

dark, sections were visualized using a fluorescence microscope

(Axio Imager A1, Zeiss, Jena, Germany).

2.8. Morphological analysis of the multiphasic scaffoldTriphasic constructs seeded with zonal chondrocytes (alginate)

and osteoblasts (biphasic PCL scaffold) were cultured in basal

medium supplemented with 10% FBS for one week prior to ima-

ging for morphological analysis (figure 1b). Triphasic constructs

were fixed in 4% PFA for 30 min and immersed in PBS/5 mM

CaCl2 solution to be imaged using the stereo microscope

(DMS1000, Leica, Australia). PFA-fixed constructs stained with

rhodamine–phalloidin and 40,6-diamidino-2-phenylindole

(DAPI) (all Invitrogen) in 5 mM CaCl2 in PBS solution were

imaged using a Nikon A1R confocal laser scanning microscope

system (Nikon Corp., Tokyo, Japan). Samples fixed in 3% glutar-

aldehyde in 0.1 M cacodylate buffer (pH 7.4) were imaged

without dehydration processes using Hitachi analytical table top

microscope TM3000 (Hitachi, Tokyo, Japan) operating at 5 kV.

2.9. Micro-computed tomographyOsteochondral explants were fixed in 4% PFA and stored in 0.1%

sodium azide in PBS at 48C prior to imaging. In order to visualize

the mineralized tissue, fixed osteochondral explants were scanned

in a mCT40 (Scanco Medical, Bruttisellen, Switzerland) at a

resolution of 12 mm, a voltage of 45 kVp and a current of

177 mA. The volume of newly formed bone within the bone com-

partment of the osteochondral construct was measured by the

micro-computed tomography (mCT) software using a greyscale

threshold of 220. For this experiment, the bovine bone was manu-

ally excluded for considering uniquely the newly formed bone

matrix (cell-free constructs: n ¼ 4; NC and Comp constructs: n ¼14). Three-dimensional images of the scaffolds were reconstructed

from the scans by the mCT system software package.

2.10. Equilibrium partitioning of an ionic contrastagent – micro-computed tomography

In order to visualize the GAG distribution in the cartilage and

alginate components of the explants, equilibrium partitioning

of an ionic contrast agent–mCT (EPIC-mCT) analysis was used,

according to the protocol described by Benders et al. [29].

Explants were incubated in 1 ml of ionic contrast agent solution

(40% ioxaglate (Hexabrix, Mallingckrodt, Hazelwood, MO,

USA)/60% PBS) for 16 h at 48C and scanned using mCT40

(Scanco Medical) at 45 kVp/177 mA. Scanned images were

analysed using Scanco mCT software (Scanco Medical).

Attenuation threshold levels were set between 1500 and 3500

Hounsfield units (HU) for all samples analysed. Gaussian filter

was used with Sigma of 1.2 and a support of 2. Three cross-

sectional images were taken at varying locations. As samples

immersed in the contrast agent may not be suitable for other ana-

lyses, EPIC-mCT was first performed using limited sample

numbers (n ¼ 2 per group). Based on the result of the first analy-

sis showing small differences between the groups, the rest of the

samples were allocated for other analysis.

2.11. Histological and immunohistochemical analysis ofosteochondral explants

Fixed osteochondral explants were decalcified in 5% formic acid

at room temperature for 14 days under constant agitation. Formic

acid was changed with fresh solution every other day. Following

the decalcification process, explants were embedded in paraffin

blocks and sectioned at 5 mm thickness. Haematoxylin and

eosin (H/E) staining was carried out using a Leica ST5010 Auto-

stainer (Leica Microsystems Pty Ltd, North Ryde, Australia).

Sections were also stained with 0.1% w/v Safranin-O for

10 min and counter stained with 0.05% w/v fast green and

Weigert’s iron haematoxylin for 5 and 10 min, respectively.

0.4

0.3

0.2

0.1

0S MD

S MD

S

** **

MD

12

8

4

0

200

150

100

50

0

DN

A (

mg)/

cons

truc

tG

AG

(mg

)/co

nstr

uct

E (

kPa)

S

MD

MD

S

NC

NC

NCCol II Col I

NC

Comp Comp

Comp Comp

NC

NC

Comp

NCComp

Comp

(b)

(c)

(d )

(a)

Figure 2. Zonal construct analysis after 12 weeks of pre-culture and one week of compressive loading (Comp) or free-swelling (NC) culture. (a) S and MD constructsexpressed both collagen types I and II during the long-term culture. (b) DNA and (c) GAG content in constructs did not vary significantly regardless of zones orloading conditions. MD constructs were significantly stiffer compared with the S constructs under free-swelling conditions, and one-week compression decreased thestiffness in MD zone constructs (d ). **p , 0.01. Scale bars, 100 mm.

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Stained sections were dehydrated in graded ethanol baths and

xylene and mounted using Eukitt (all Sigma). Immunohistochem-

ical analyses of the explants for collagen type I (Col I, dilution 1 :

500, I-8H5, MP Biomedicals), collagen type II (Col II, dilution 1

: 200, II-II6B3, DSHB), von Willebrand factor (vWF, dilution 1 :

3\0, Millipore AB7356) and alkaline phosphatase (ALP, dilution

1 : 500, B4–78, DSHB) were carried out using a commercially

available kit (VectaStain ABC kit, Vector Labs Inc., Burlingame,

CA, USA) using the instructions provided by the manufacturer.

Prior to staining for Col I, Col II and ALP, sections were soaked

in 10 mM sodium citrate/0.05% Tween 20 buffer at pH 6.0 and

heated in a pressure cooker for 4 min at 948C. For vWF, sections

were incubated with few drops of proteinase K (Dako, Den-

mark). After rinsing in PBS, sections were treated with 3%

H2O2 for 10 min and blocked with 2% normal horse serum sup-

plied in the kit for 1 h. Sections were then incubated with

primary antibody solution diluted in the blocking buffer for

1 h followed by two 5 min washes in PBS bath. Sections were

further incubated in biotinylated secondary antibody solution

and Vectastain ABC reagent provided in the kit for 30 min

each. Bound peroxidase was visualized using ImmPACT DAB

peroxidase substrate kit (Vector Laboratories, USA), and

images were captured using a Leica SCN400 Slide Scanner

(Leica Biosystems, Wetzlar, Germany). Decalcified human

osteochondral sections were used as controls for histological

and immunohistochemical staining.

2.12. Osteocyte etchingOsteochondral samples fixed in 4% PFA were dehydrated in

graded series of ethanol and infiltrated using ethanol/resin sol-

ution with increasing resin concentration (from 30% v/v resin to

100% resin) for 4 days (Tecknovit 7200, Heraeus Kulzer, Germany)

in an EXAKT 510 dehydration and infiltration system. Resin was

then cured using an EXAKT 520 light polymerization system

(EXAKT, Germany). Once cured, resin blocks were ground using

an EXAKT 400 CS micro grinding system to expose the samples

and subsequently cut using an EXAKT 300 cutting system

equipped with a diamond saw. Resin blocks were polished using

400, 600 and 1200 grit sandpapers and a final polish achieved

with 1.0 alpha alumina powder polishing using a soft cloth polish-

ing wheel. Mirror-finished block faces were etched with 37%

phosphoric acid for 2–5 s and washed with 12.5% sodium hypo-

chlorite for 5 min followed by a brief rinse in deionized water

[30]. Acid-etched blocks were sputter-coated with gold (Leica EM

SCN005) and visualized using an FEI Quanta 200 environmental

scanning electron microscope operating at 10 kV.

2.13. StatisticsSPSS (v. 19.0) was used to perform the statistical analysis. Data are

presented as mean+ s.d. Two-factor ANOVA and Fisher’s least sig-

nificant difference post hoc tests were used to determine the effects of

zone and pre-culture on GAG, DNA and compressive modulus in

figure 2b (n ¼ 6). In all tests, p , 0.05 was considered significant.

3. Results3.1. Analysis of zonal (surface and middle-deep)

constructs following long-term culture andcompressive stimulation

Following 12 weeks of in vitro culture under chondrogenic

conditions, S and MD constructs stained intensely for

atte

nuat

ion

(HU

)

–1000 10 938(HU)

–1000 10 938(HU)

–1000 10 938(HU)

cell-free

compressed

non-compressed

3500

1500

(b)

(a)

(c)

Figure 3. EPIC-mCT images and histograms of attenuation in the cartilaginous compartments (dotted area) following in vivo implantation. Lower attenuation levelsindicate higher GAG content and high attenuation levels indicate bone or tissues with low GAG content. (a) Cell-free alginate gel, (b) alginate gel subjected to oneweek of compression and (c) non-loaded gel implanted within the cartilaginous compartment had similar attenuation levels between the different groups, anddepth-dependent zonal variations were not observed. Bone-like attenuation patterns were visible in the outer rims of the bovine cartilage in all explants.

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collagen type II (Col II) around chondrocytes but limited

staining was observed in spaces further away from the cells

(figure 2a). Collagen type I (Col I) was also expressed in

some zonal chondrocytes and was less intense compared

with Col II. Both Col II and I stained similarly between

groups that were compressed for one week (Comp) and

those that were NC. DNA and GAG levels did not vary

significantly between groups regardless of zones or loading

conditions (figure 2b,c). Unconfined compression tests

revealed that MD constructs were significantly more stiff

compared with the S constructs under free-swelling con-

ditions (figure 2d; p ¼ 0.002). Compressive stimulation

decreased stiffness of MD constructs but not in S constructs

( p ¼ 0.009).

3.2. Triphasic scaffold assemblyAlginate discs seeded with surface and middle-deep

chondrocytes were well integrated, with visibly smooth tran-

sition line, as shown under the stereo microscope (figure 1b).

The PCL-FDM scaffold and the melt-electrospun PCL mesh

were also well integrated. Scanning electron microscopy

images show that top portions of the thin mesh filaments

are fused with the larger FDM scaffold fibres, which had

been partially melted by the heat treatment (figure 1b). The

interface between the zonal alginate construct and the PCL

scaffolds was relatively more fragile than the other interfaces,

yet was able to withstand manual handling using forceps.

Partial de-cross-linking of the alginate and press-fitting it

on top of the porous FDM scaffold resulted in alginate infil-

tration through the pores of the PCL scaffold and the

topmost layer of the PCL scaffold was partially encapsulated

in alginate gel (figure 1b). Fibrin glue used for the rh-BMP-7

delivery also served to hold the construct in place within the

osteochondral defect (figure 1c), and construct loosening did

not occur during the implantation procedures (figure 1d ).

3.3. Analysis of osteochondral constructs following invivo implantation: equilibrium partitioning of anionic contrast agent – micro-computed tomographyand micro-computed tomography

High degree of attenuation in the EPIC-mCT images has

been reported to show low GAG concentration in cartilage

[29]. The upper portion of the osseous compartment and

the inner parts of the cartilage compartment of the construct

had lower attenuation levels (figure 3). Zonal differences in

attenuation levels between the S and MD alginate layers

were not apparent. Attenuation level distributions were

also similar in cell-free (figure 3a) and cell-containing

alginate layers cultured with (figure 3b) or without com-

pression (figure 3c). The outer edge of the bovine

cartilage, along with the bovine bone had high attenuation

in the EPIC-mCT images, which is most likely owing to cal-

cification of the cartilage in these regions. This is apparent

from the three-dimensional mCT images of explants not

stained with Hexabrix, which show that the outer rims of

the bovine cartilage and the upper portion of the bovine

bone had substantial mineralization in most explants

(figure 4). Surprisingly, there was limited mineralization in

the PCL compartment with or without the pre-seeded osteo-

blasts (figure 4e) and no significance differences were found

between the groups (figure 4i).

3.4. Analysis of cartilage compartment: histology andimmunohistochemistry

The intensity of Safranin-O within the bovine tissue was the

strongest in the hypertrophic zone of the cartilage near the

bone and the newly mineralized regions on the outer edge

of the cartilage (figure 5a,b). These areas stained faintly of

collagen type II and had distinct boundaries (figure 5c,d ).

(b)

(a)

(c) (g)

(h)(d )

(e)

( f )

2.5

2.0

1.5

1.0

0.5

0

cell-free

compressed

non-compressed

bone

vol

ume

(mm

3 )

(i)

Figure 4. mCT images showing mineralized tissues in the scaffold. Explants of the triphasic scaffold inserted in bovine core, which had cell-free (osteoblast) (a,e) and osteo-blast-seeded bone compartments ( patient 1: b,f; patient 2: c,g; patient 3: d,h), had little bone formation in the osseous compartment. Explants that contained alginate gelssubjected to a one-week compressive loading prior implantation are shown in (e – h), and those that had non-loaded alginates are shown in (a – d ). Mineralization was visibleon the outer rims of the cartilage in all groups including the cell-free controls. (i) Bone volume within the bovine osteochondral defect as measured by mCT. There was nostatistical difference among the three groups (cell-free n¼ 4, Comp and NC n ¼ 14). (Online version in colour.)

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Layered alginate constructs were largely intact in constructs

pre-seeded with cells compared with those that were cell-

free at the time of implantation (figure 6a– i). A thin line of

cells was found in some areas in between the S and MD

layers. Cells in the alginate appeared either as isolated

cells or were in large aggregates. Cell aggregates stained

faintly of collagen type II in the centre. Cell-free alginate

gels were fully infiltrated with fibrous tissues and many

multinucleated cells were found in between the alginate

debris (figure 6j–u).

3.5. Analysis of bone compartment: histology,immunohistochemistry and osteocyte etching

Sections stained for vWF indicated a limited blood vessel net-

work within the osseous construct while there were many

(b)(a) (c) (d )

bovinebone

bovinebone

bovinecartilagebovine

cartilage

triphasic construct triphasic construct

2m

m

2m

m

Figure 5. (a,b) Safranin-O and (c,d ) collagen type II-stained images of bovine cartilage tissue following in vivo implantation. Areas of the cartilage corresponding tothe mineralized regions are indicated by the open arrow. (b,d) Scale bars, 200 mm. (Online version in colour.)

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blood vessels found within the bovine bone (figure 7a,b, black

arrowheads). Scanning electron micrographs of acid-etched

explants also show that osteocytes within the bovine bone had

well-preserved processes, which were extensively interconnected

with each other (figure 7c,d, black arrowheads) although those

within approximately 100 mm of inner and outer edges of the

bone had relatively diminished interconnectivity and some

empty lacunae, possibly from heat or mechanically induced

cell injury during the osteochondral plug preparation. The

upper regions of the bovine bone, which were also densely

mineralized according to the mCT analysis, had numerous

patches, which stained strongly with Safranin-O (figure 7e,white arrowheads). These regions of bone also contained many

cells, which stained positive for ALP, as indicated by the black

arrowhead in figure 7f. Lower parts of the bone also exhibited

Safranin-O (figure 7g, white arrowheads)-stained regions and

ALP-positive cells (figure 7h, black arrowhead), but to a lesser

degree. Although the fibrous interstitial tissue stained intensively

for ALP in both the top and bottom portion of the bovine osteo-

chondral plug (figure 7f,h), there was a clear difference within the

bovine bone with the top portion having a large number of ALP-

positive stained cells as opposed to the bottom portion where

only a very few cells were positively stained.

3.6. Fibrous tissue infiltrationFibrous tissue infiltration occurred throughout the osteochondral

explants in all samples. In the bovine cartilage, fibrous tissues

were found to penetrate towards the periphery of the mineraliz-

ing areas (figure 8a,b,e,f,i,j) from the both outer and inner edge

of the bovine cartilage. Fibrous tissue infiltration also occurred

from the top portion of the cartilage and immediately below

the mineralized regions (figure 8c,g,k). Rat fibroblasts also satu-

rated the electrospun mesh and formed a cellular bridge

between the mesh and cartilage (figure 8c,g,k). Fibrous tissues

fully surrounded the PCL-FDM/melt-electrospun scaffold in

the osseous compartment, and they were also present throughout

the bovine bone (figure 8d,h,l ). Macrophage-like multinucleated

cells were visible in some areas of the fibrous tissue, which may

indicate inflammatory responses.

4. DiscussionPreclinical animal testing is an important step towards the

translation of newly developed tissue-engineering concepts

from bench to bedside. Immunodeficient rodents are widely

used for xenograft-based studies and have the benefit of low

cost and manageability. Subcutaneous implantation in severe

combined immunodeficiency mouse or rat is a well-established

method of studying human tissue development and has been

used to study the development of tumours [31], bone [32]

and cartilage [33]. Bovine osteochondral cores have been

used in the past to study cartilage defect repair in vitro [34],

as they have comparable cartilage thickness to humans. Com-

bining the use of bovine osteochondral defect plugs and

subcutaneous implantation strategies is a new concept that

has not been well characterized. In this study, we developed

a hybrid in vivo animal model to implant multiphasic osteo-

chondral constructs and evaluated the outcomes after

12 weeks. Our results indicate that our hybrid in vivo defect

model requires further optimization to support robust bone

and cartilage formation.

Multiphasic scaffold design is a growing trend in osteo-

chondral tissue engineering, with a recent increase in the

number of studies exploring the biphasic concepts. There

are advantages in using multiphasic designs over monopha-

sic constructs for osteochondral repair. For instance, scaffold

types and growth factor additives can be optimized for the

development of cartilage and bone separately, and the

option of post-assembly allows for chondrogenic and osteo-

genic pre-culture prior to the in vivo implantation [35].

However, unlike monophasic constructs, designing the carti-

lage–bone interface in multiphasic tissue graft is a major

challenge. Although the presence of bovine defect plug

offers a degree of protection from external mechanical stres-

ses, histological analysis of the implanted constructs shows

that the alginate constructs had been separated from the

PCL scaffolds in some explants possibly owing to gradual

weakening of the interface region (figure 6a– i).Recapitulation of zonal architecture and mechanical prop-

erties similar to native articular cartilage remain important

goals in functional regeneration of cartilage. As a result,

recent developments in cartilage tissue engineering show a

growing number of investigators attempting to incorporate

zonal architecture [36] and mechanical pre-conditioning [37]

to further improve the functionality of the cartilage construct

in vitro. In our study, one week of mechanical loading did not

significantly affect collagen types I, II, total DNA and GAG

levels and using S and MD zone chondrocytes to fabri-

cate layered constructs did not result in zonal differences

bovinealginate

alginatealginate

fibrous tissue

fibrous tissue

fibrous tissue

fibrous tissue

fibrous tissuefibrous tissue

cartilage

bovinecartilage

bovinecartilage

non-compressed

haem

atox

ylin

and

eos

inco

llage

n ty

pe I

colla

gen

type

II

compressed cell-free

(b)(a) (c)

(d ) (e) ( f )

(g) (h) (i)

( j) (k) (l)

(m) (n) (o)

(p) (q) (r)

(s) (t) (u)

Figure 6. Histological and immunohistochemical images of explant cartilaginous compartment. While explants implanted with both NC (a,d,g,j,m,p,s) and Comp(b,e,h,k,n,q,t) alginate gels had retained much of the alginate constructs during the 12-week in vivo culture, those in the cell-free group had little alginate left(c,f,i,l,o,r,u). While S and MD alginate gels remained intact in the cell-seeded groups, fibrous tissue infiltration was visible in the interface between the two gels(arrows). Cells found within the alginate compartment in all groups formed dense aggregates but did not stain strongly for either collagen type I ( j – o) or II ( p – u).Scale bars: (a – c), 1 mm; (d – f, j – l, p – r), 200 mm; (g – j, m – o, s – u), 50 mm. (Online version in colour.)

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(figure 2). Not surprisingly, EPIC-mCT images of the explants

also show that we were not able to achieve zone-specific

differences in the GAG distribution in constructs with or

without the mechanical loading (figure 3). Compared to the

12 weeks of culture, one week is a relatively short period of

time, and it is possible that mechanical loading should be

applied earlier and for a longer duration for optimal results.

Also, as indicated by others, expanded chondrocyte sub-

populations gradually lose zonal phenotypes in vitro [38]

and may require zone-specific differentiation strategies. In

future studies, improvements in zonal cartilage formation

may be achieved by applying different types of mechanical

load (e.g. shear stress, hydrostatic pressure) to better

represent zone-specific physiological load.

One of the benefits of implanting osteochondral constructs

within the osteochondral defect is that resident chondrocytes

and osteoblasts in the surrounding osteochondral tissues may

influence the integration and the differentiation processes of

(b)(a)

(c)

(g) (h)

(d )

(e) ( f )

bovine bone

fibrous tissue

fibrous tissuePCL FDM

fibre

Figure 7. Representative imaging of the bovine bone across the different groups. vWF staining shows well-developed blood vessels in and around the bovine bone(black arrowhead) (a), while little to no defined vWF staining is visible within the osseous scaffold within the defect (b) from a NC sample. Osteocyte etching revealswell-preserved cells in the bone that are in close proximity to the infiltrated fibrous tissues (black arrowhead) (c) and those that are well within the bone (d ) from aNC sample. Representative histological images from a NC sample, which indicate areas within the bone staining intensely with Safranin-O (top (e), bottom (g), whitearrowhead). Sections of a compressed sample stained for ALP show depth-dependent staining patterns with more cells staining for ALP near the top portion of thebone ( f ) compared with those in the bottom (h) (black arrowheads). Scale bars: (a,b,e – h), 200 mm; (c,d ), 50 mm. (Online version in colour.)

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the immature construct. Our analysis of the mCT images shows

that some parts of the bovine cartilage had gone through

hypertrophy, especially at the outer edges of the bovine

cartilage (figure 4). There was a clear boundary around the

mineralized areas of the cartilage, which stained intensely

with Safranin-O, but had little to no stain for collagen types I

and II (figure 5). This observation is comparable to the cal-

cification process of the growth plate cartilage where

concentration of aggregating proteoglycans and collagen

type II in the growth plate cartilage is at its highest immedia-

tely before hypertrophy, followed by a dramatic decrease, as

the tissue gets mineralized [39]. Sharp transitions of proteo-

glycans and collagen content seen near the edges of the

mineralized cartilage may be indicative of an ongoing endo-

chondral ossification-like process. Factors that may have

contributed to cartilage calcification are chondrocyte hyper-

trophy and apoptosis, and the presence of inflammatory

cytokines and rh-BMP-7 [40].

Bone morphogenic protein-7 is well known for its ability

to stimulate bone formation in vivo [41], but robust bone

formation and long-term survival are also dependent on suc-

cessful angiogenesis. In this study, the use of rh-BMP-7 in the

implanted osseous constructs did not sufficiently augment

bone development even though there was prominent miner-

alization in the bovine cartilage. Studies suggest that vascular

invasion and mineralization can occur in tissue-engineered

cartilage following ectopic implantation in mouse, especially

when they are developed using pre-differentiated human

mesenchymal stem cells [42] or expanded chondrocytes

[43]. These studies also show that constructs developed

(b)(a)

(c)

(d )

(e)

(g)

(h)

( j)

(k)

(i)

(l)

( f )triphasic scaffold triphasic scaffold

triphasic scaffold

PCL FDMscaffold

PCL FDM scaffold

PCL FDM scaffold

PCL FDMscaffold

PCL FDM scaffold

PCL FDMscaffold

bovinetissue

bovinetissue

bovinetissue

bovinebone

bovinebone

bovinebonemelt electrospun

scaffold

melt electrospunscaffold

melt electrospun scaffoldmelt electrospun scaffold

melt electrospunscaffold

melt electrospun scaffold

Figure 8. Haematoxylin-/eosin-stained sections of osteochondral explants. (a – d ) NC, (e – h) Comp and (i – l ) cell-free. Fibrous tissue infiltration was seen in both theinner and outer edges near the lower portion of the bovine cartilage in all explants (a,e,i, and arrowheads in b,f,j ). Fibrous tissue infiltration was also seen in the upper partof the cartilage and they were seen primarily from the inner edges of the cartilage (arrowheads in c,g,k). Fibrous tissues also fully infiltrated the bovine bone, PCL-FDMscaffolds and the PCL mesh (a,e,i,d,h,l ). Scale bars: (a,e,i), 1 mm; (b – d,f – h,j – l ), 200 mm. (Online version in colour.)

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from fully differentiated (i.e. freshly isolated) chondrocytes

tend to resist vascular invasion and mineralization. However,

what was apparent in our study was that most pronounced

mineralization occurred mainly in the bovine cartilage, rather

than in the engineered cartilage containing the expanded chon-

drocytes. A possible reason for this, other than rh-BMP-7, may

be the close proximity of the mineralized parts of the bovine

cartilage to the host vasculature, increasing its vulnerability

to hypertrophy.

On the other hand, the lack of bone formation in the

osseous compartment may be owing to insufficient vascu-

lar ingrowth. Sections stained for vWF indicate higher

concentration of blood vessels within the bovine bone

compared with the osseous compartment of the construct

(figure 7a,b). Highly vascularized areas of the bovine bone

also had well-preserved osteocytes (figure 7c,d ), and in

these areas histological images show a number of small

defined areas which stained intensely with Safranin-O, an

indicator of cartilage-like tissue formation. Cells that stained

for ALP were found in larger numbers near the top portion

of the bovine bone, coinciding with the higher number of

Safranin-O-stained areas near the top portion of the bone

(figure 7e,f ). These observations show that the role of angio-

genesis in bone formation is prominent and that it often

dictates the success of bone regeneration.

It is possible that dense bovine bone may have acted as a

barrier for vascular infiltration. Although the bovine bone

itself was vascularized, spaces within the bone were occupied

by fibrous tissues, which together with the dense mineralized

matrix can be a physical barrier to vascular ingrowth.

Delayed or compromised vascular infiltration may affect the

construct development in two ways. Firstly, prolonged depri-

vation of oxygen and nutrients would have decreased the

viability of the pre-seeded osteoblasts, rendering them inef-

fective in initiating bone regeneration. While longer in vitroosteogenic differentiation of osteoblasts on the PCL scaffolds

may help to kick start mineralization, the vascularization

issue will likely remain an issue for post-implantation bone

regeneration. Secondly, the delivery method of rh-BMP-7 to

the osseous compartment was through fibrin glue [44], with

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the resorption time frame of two weeks during an in vivoimplantation [45]. If rh-BMP-7 leached out of the defect site

prior to sufficient cellular and vascular infiltration, it may

have induced bone formation elsewhere, with limited

rh-BMP-7-induced bone formation within the osseous com-

partment. Synthetic oxygen carrier-enriched hydrogels as

rh-BMP-7 delivery vehicles may be considered as an alterna-

tive to fibrin glue, as they have been shown to help bone

regeneration [46]. The use of hydrogel-coated microspheres

to deliver VEGF (gelatin hydrogel) prior to rh-BMP-7

(PLGA) has been reported to improve ectopic bone formation

in rat through improved vascular network [47]. Increasing the

duration of the release profile of rh-BMP-7 and combining

other growth factors, such as VEGF, may help better bone

regeneration in the multiphasic constructs in future studies.

While the osteochondral plugs provided structural sup-

port for the implanted constructs, they did not shield them

from fibrous tissue infiltration. Not only was fibrous tissue

infiltration observed in the bovine bone and the osseous

compartment of the triphasic scaffold, fibrous tissue pen-

etration was visible within the non-mineralized areas of the

bovine cartilage and alginate constructs (figure 8a– l ). A fibrous

tissue layer had bridged the alginate and bovine cartilage

together and had infiltrated into the alginate compartment

(figure 6d–o). They also filled the interface region between

the two alginate layers (figure 6d– i) and aggregated into

small spheroids in some areas within the alginate. Interestingly,

the morphology of the alginate constructs and the degree of rat

fibroblast infiltration varied between the chondrocyte-seeded

and the cell-free groups. Chondrocyte-seeded alginate con-

structs retained their overall shape while cell-free alginate

constructs did not. Large multinucleated cells were also visi-

ble around the remaining alginate in the cell-free constructs

(figure 6d–o). While it is not clear as to why the chondrocyte-

seeded alginate gels fared better against fibrous tissue infiltra-

tion, other groups have also noted that constructs seeded with

cells survive better when implanted in osteochondral defects

compared with the acelluar constructs [48].

A limiting factor in the presented model is the lack of

mechanical stress, which routinely occurs in the knee joint. It

is evident that mechanical stimulation through compressive,

tensile and hydrostatic loading improves matrix production

in both chondrocytes and osteoblasts [49–51]. Mechanical

stimulation also appears to influence osteoblast and osteoclast

activity and encourages mineralization [52,53]. While it will be

difficult to apply physiological loads in this hybrid in vivomodel, it does allow for optimization of several other factors

important in osteochondral repair, such as cell source and bio-

material type. The repair tissue in the hybrid in vivo defect

model may have arisen from a mixture of cells from three

different species (human, bovine and rat). The use of tissues

from different species may actually be advantageous, by iden-

tifying the source of the cells and tissues using species-specific

antibodies. Nonetheless, the use of three different species is not

a requirement, and using osteochondral cores from normal or

osteoarthritic human donors may offer the potential to study

the effects of the osteoarthritic environment on cartilage

repair, but they are difficult to obtain frequently or in large

quantities. Finally, the model described here employed multi-

phasic constructs with hydrogels and composite scaffolds of

different scales and fabrication techniques but could be used

with virtually any biomaterial and scaffold repair approach.

Taken together, this study provides a proof-of-concept for a

novel in vivo osteochondral defect model. With further

optimization, the presented hybrid in vivo model may help

address the growing need for a cost-effective way to screen

osteochondral repair strategies before moving to large-animal

preclinical trials.

Acknowledgement. Antibodies II-II6B3 (developed by ThomasF. Linsenmayer) and B4–78 (developed by Jerry A. Katzmann)were obtained from the Developmental Studies Hybridoma Bankdeveloped under the auspices of the NICHD and maintained bythe University of Iowa, Department of Biology, Iowa City, IA52242, USA.

Funding statement. The research leading to these results has receivedfinancial support from the European Union’s Seventh FrameworkProgramme (FP7/2007–2013) under grant agreement no. 309962(project HydroZONES) and National Health and Medical ResearchCouncil Australian–European Union Health Research CollaborationAPP1067108. Additionally, T.K. and D.W.H. were funded throughARC Future Fellowships. The authors have no conflict of interestwith regard to the work.

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