Neovascularization and free microsurgical transfer of in vitro cartilage-engineered constructs

NEOVASCULARIZATION AND FREE MICROSURGICAL TRANSFEROF IN VITRO CARTILAGE-ENGINEERED CONSTRUCTS

NGUYEN THE HOANG, Ph.D., M.D.,1,2,3* CHRISTOPH HOEHNKE, Ph.D., M.D.,2 PHAM THU HIEN, M.D.,3

VERONIKA MANDLIK, M.D. (student),3 ACHIM FEUCHT, M.D. (student),3 and RAINER STAUDENMAIER, Ph.D., M.D.3

Cartilage tissue engineering shows to have tremendous potential for the reconstruction of three-dimensional cartilage defects. To ensuresurvival, shape, and function, in vitro cartilage-engineered constructs must be revascularized. This article presents an effective method forneovascularization and free microsurgical transfer of these in vitro constructs. Twelve female Chinchilla Bastard rabbits were used. Carti-lage-engineered constructs were created by isolating chondrocytes from auricular biopsies, amplifying in monolayer culture, and then seed-ing them onto polycaprolactone scaffolds. In each prefabricated skin flap, three in vitro cartilage-engineered constructs (2 3 2 3 0.5 cm)and one construct without cells (served as the control) were implanted beneath an 8 3 15 cm random-pattern skin flap, neovascularizedby implantation of an arteriovenous vascular pedicle with maximal blood flow. Six weeks later, the neovascularized flaps with embeddedcartilage-engineered constructs were completely removed based on the newly implanted vascular pedicle, and then freely retransferredinto position using microsurgery. Macroscopic observation, selective microangiography, histology, and immunohistochemistry were per-formed to determine the construct vitality, neovascularization, and new cartilage formation. The results showed that all neovascularizedskin flaps with embedded constructs were successfully free-transferred as free flaps. The implanted constructs were well integrated andprotected within the flap. All constructs were well neovascularized and showed histologically stability in both size and form. Immunohistol-ogy showed the existence of cartilage-like tissue with extracellular matrix neosynthesis. VVC 2008 Wiley-Liss, Inc. Microsurgery 29:52–61,2009.

Tissue engineering can provide a promising method for

repairing or replacing any tissue in the human body that

is injured or damaged as a result of disease or trauma. In

spite of technological advances and remarkable scientific

progress in recent years, there are, however, very few

clinical applications of tissue engineering reported in the

literature. Challenges relating to the clinical use of tissue

engineering are listed as follows: (a) problems associated

with cell expansion, (b) challenges relating to cell sur-

vival and function after seeding onto scaffolds, as well as

necrosis of tissue-engineered constructs due to vascular

disruption following implantation or transplantation,1 and

(c) difficulties associated with optimal scaffolds for cell

seeding (e.g., biocompatibility and biodegradation, elas-

ticity and mechanical stiffness, ability to allow nutrient

diffusion, appropriate environment for cell adhesion,

growth, differentiation and proliferation, etc.).2

Although numerous publications were presented in the

literature relating to the use of newly developed proce-

dures for optimizing the cartilage tissue engineering

method for future clinical applications (e.g., bioreactor

systems,3,4 cell type,5,6 growth factors,7–9 etc.), studies on

revascularization of in vitro cartilage-engineered con-

structs to ensure their in vivo survival, function, and

shape—the ultimate goal of the procedure—were, how-

ever, only rarely investigated and reported on.10,11

Although cartilage is an avascular tissue consisting of

only chondrocytes, which are embedded in a matrix com-

posed of collagen and proteoglycan,2,12 newly cartilage-

engineered constructs with a large volume of expended

cells usually require a hyperoxic environment for their

growth and proliferation.10 Disruption of blood and nutri-

ent supply results in cell death and unavoidable necrosis

of the cartilage-engineered construct with subsequent loss

of shape and function.1,11

In 2004, Staudenmaier et al.10 reported preliminary

experimental results of flap prefabrication and prelamina-

tion with tissue-engineered cartilage using non-woven

fleece scaffolds HYAFF1 (hyaluronic-axid derivative,

Biopolymers, Abano Terme, Italy), which were placed in

different regions of the rabbit body including the prefab-

ricated flap, subcutaneous abdominal wall, and in an

intramuscular pocket of the thigh. Neovascularization and

neocartilage formation in the constructs were analyzed by

angiography, histology, and immunohistology. Despite

the natural polymer fiber, HYAFF1 has been identified

by this study as an effective scaffold material based on

excellent biocompatibility and shape-conforming proper-

ties; however, it lacks the mechanical strength and ability

1Department of Hand Surgery and Microsurgery, Institute of Trauma andOrthopedics, Central University Hospital 108, Hanoi, Vietnam2Department of Plastic Surgery, University Hospital ‘‘rechts der Isar’’, Techni-cal University of Munich, Germany3ENT Department, University Hospital ‘‘rechts der Isar’’, Technical Universityof Munich, Germany

Grant sponsor: Bayerische Forschungsstiftung (FORTEPRO); Grant number:Az. 442/01.

*Correspondence to: Nguyen The Hoang, Ph.D., M.D., Khoa B1-2, VienChan Thuong Chinh Hinh, Benh Vien 108, So 1-Tran Hung Dao, Hanoi,Vietnam. E-mail: [email protected]

Received 2 May 2008; Accepted 31 July 2008

Published online 22 October 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/micr.20565

VVC 2008 Wiley-Liss, Inc.

to withstand in vivo mechanical load-bearing, and there-

fore is not ideal for cartilage defect reconstruction.

Recently in a rat model, Neumeister et al.11 reported on

vascularized tissue-engineered ears by implantation of

in vitro cultured chondrocytes into molded fibrous capsu-

les appearing around an implanted human ear-shaped sili-

cone, neovascularized by transplantation of a femoral

vessel bundle onto the block. The development of new

cartilage tissue was determined by histology.

With respect to clinical applications, we hypothesized

that the survival and transplantation of tissue-engineered

constructs should be very easy and effective if they were

neovascularized and transferred as an axial free flap

based on a reliable vascular pedicle. This study was

designed to investigate the ability of combining flap pre-

fabrication and tissue engineering constructs as well as

neovascularization and free microsurgical transplantation

of combined skin-cartilage-construct-engineered flaps by

means of implanting cartilage-engineered constructs

within a prefabricated skin flap.

MATERIALS AND METHODS

Twelve adult female Chinchilla Bastard rabbits

weighing from 3 to 4 kg were investigated in this study.

They were housed in accordance with the European

Directive for the Care and Use of Lab Animals (Reg.

Obb. AZ 211-2531-38/95). Anesthesia was induced by in-

travenous infusions of a mixture of ketamin 40 mg/kg

and xylazin 4 mg/kg (i.v.). All operations were performed

under sterile conditions. Three procedures were per-

formed on each animal including: (1) in vitro fabrication

of cartilage-engineered constructs using polycaprolactone

(PCL)-based polyurethane scaffolds, (2) in vivo neovas-

cularization by mean of implantation within a prefabri-

cated flap, and (3) microsurgical transplantation of these

neovascularized cartilage-engineered constructs.

In Vitro Construct Fabrication

The auricular cartilage harvested from the left ear of

Chinchilla Bastard rabbits was used for the investigation.

The cartilage specimen was washed in saline and minced

into 1 3 1 mm pieces in a petri dish. The chondrocytes

were enzymatically isolated during 6 hours incubation at

378C in a collagenase solution (1,108 U/ml) and resus-

pended in Dulbecco’s modified Eagle medium (Sigma,

Berlin, Germany) containing 4.5 g/l glucose, 548 mg/l

glutamine, 10% fetal calf serum (Life Technologies,

Karlsruhe, Germany), 50 U/ml penicillin (Biochrom, Ber-

lin, Germany), 1 mM nonessential amino acids (Bio-

chrom, Berlin, Germany), and 0.4 mM proline (Sigma,

Berlin, Germany). For expansion, the chondrocytes were

cultured under conventional monolayer culture conditions

at 378C, 5% CO2 for 2 weeks. The cell suspension was

filtered using a 70 lm filter, pelleted by centrifuging at

1,500 rpm for 10 minutes, and washed three times with

phosphate buffered saline (PBS; Grosshadern, Munich,

Germany).

For cell-construct fabrication, cultured cells were sus-

pended in 20 ll fibrinogen solution and mixed with the

same volume of thrombin solution. Subsequently, the

cell-fibrin suspension was seeded onto 2 3 2 3 0.5 cm

samples of PCL-based polyurethane scaffolds (PolyMate-

rial AG, Kaufbeuren, Germany) at a concentration of

�20 3 106 cells per cm3. After an adhesion period of 5

hours the medium, supplemented with 50 lg/ml ascorbic

acid (Merck, Darmstadt, Germany), was renewed and

then subsequently replaced every 2–3 days. For each ani-

mal in this study, three cell-biomaterial constructs were

fabricated and further cultured for 1 week before in vivo

implantation. In addition, a construct without cells with

the same design and dimension served as control was

also prepared for in vivo implantation.

In Vivo Neovascularization and Free Transfer

of Cartilage-Engineered Constructs

Cartilage-engineered constructs and control constructs

were first neovascularized by means of implantation

within a prefabricated skin flap and next free-transferred

based on the newly axial implanted vascular pedicle

using microsurgical techniques.

Prefabrication of a neovascularized skin flap. On

the right abdomen of Chinchilla Bastard rabbits, a ran-

dom-pattern skin flap measuring 8 3 15 cm with lateral

pedicle was dissected and lifted up (Fig. 1A). After con-

trolling hemostasis throughout this area with a bipolar

electrocautery, a silicone sheet (LPI, Perouse, 60540 Bor-

nel, France) measuring 8 3 15 3 0.25 mm was placed

on the abdominal wall and anchored with Ethilon 3/0

(Fig. 1B). This silicone functioned as a barrier to prevent

neoangiogenesis from occurring between the underlying

vascular bed and the abdominal skin flap. An incision

extending from the inguinal ligament to immediately

above the calcaneous was made to harvest the femoral

and saphena magna vascular bundle. All branching ves-

sels were ligatured or closed using a bipolar coagulator.

In the next step, the artery and vein of the distal end of

the vascular bundle were freely dissected for 3 cm and

microanastomosed end-to-end using 10-0 Nylon monofila-

ment suture (Ethicon) to create maximal blood flow

within the vascular pedicle through the newly established

arteriovenous shunt. Next, the vascular bundle was turned

in the cranial position and anchored beneath the abdomi-

nal skin flap using Vicryl suture 8/0 (Fig. 1B).

Neovascularization of the cartilage-engineered

constructs. After pedicle implantation, three cartilage-

engineered constructs (�2 3 2 3 0.5 cm) were

In Vitro Cartilage-Engineered Constructs 53

Microsurgery DOI 10.1002/micr

implanted under the skin flap, where the first construct

was placed directly onto the implanted vascular pedicle,

the second was implanted laterally, and the third

implanted medially to the vascular pedicle and fixed with

Vicryl suture 8/0. Simultaneously, an additional construct

without cells was also implanted distally of the vascular

pedicle and served as a control specimen (Fig. 1C). To

avoid negative influences from the direct contact between

silicone sheeting and cartilage-engineered constructs, a fas-

cia piece (6 3 10 cm) harvested from the affected thigh

was covered over the implanted constructs and anchored

with Vicryl 5/0. Next, all skin incisions were sutured back

into position using 3-0 Vicryl and Nylon sutures (Fig.

1D). The animals were then returned to their cages.

Free transplantation of the cartilage-engineered

constructs. Six weeks after the operation, the entire

prefabricated flap with embedded cartilage-engineered con-

structs in the same dimension and design (8 3 15 cm)

based on the newly implanted vascular pedicle were com-

pletely removed and then transferred back into place as

an axial free flap using microsurgical techniques with

Nylon 10/0 (Ethicon).

Evaluation Parameters

In this study, flap and construct viability was exam-

ined by macroscopic observation, the neovascularization

development of constructs was evaluated by selective mi-

croangiography and histology, and the neocartilage for-

mation was determined by histochemistry and immuno-

histochemistry.

Macroscopic observation. The viability of these pre-

fabricated cartilage-engineered skin flaps before and after

free microvascular transfer were evaluated macroscopi-

cally on a daily basis with respect to color, area of necro-

sis, hair growth, and wound healing over an observation

period of 2 weeks.

Selective microangiography. For selective microan-

giography of prefabricated cartilage-engineered skin flaps

after free flap transfer, the animals were reanesthetized

and heparinized intravenously (5,000 IU). The artery of

the implanted vascular pedicle was cannulated with a poly-

ethylene tube (0.9 3 25 cm) and the vascular system of

the prefabricated skin flap was irrigated with warmed

heparinized saline (378C) for 20 minutes. Next, a suspen-

sion of Micropaque 30% with Rheomacrodex 10% in the

ratio of 2:1 was injected into the artery at a constant

injection pressure of 110 mmHg for 45 minutes. Follow-

ing, the animals were given a lethal injection of Nacoren

(Pentobarbital 160 mg/kg) (Merial, Hallbergmoos, Ger-

many). The flaps were excised and pinned on cardboard

holders to perform microangiography using a Radiofluor

machine (120—TORR Philips Electronic Instrument,

Munich, Germany) and Kodak X-ray films.

Histology. Conventional histology was performed after

microangiography to evaluate viability and morphology

Figure 1. (A) Preoperative design of skin flap prefabrication; (B) overview of silicone implantation and implantation of an arteriovenous

vascular pedicle with maximal blood flow into a random-pattern abdominal skin flap; (C) simultaneous implantation of three cartilage-engi-

neered constructs and one control construct beneath the prefabricated skin flap [TE, tissue engineering]; (D) Postoperative view. [Color fig-

ure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

54 Hoang et al.


of the cartilage-engineered constructs and skin flap. Con-

structs with and without cells were marked and cut per-

pendicularly to the long axis of the vascular pedicle,

stained with Hematoxylin Eosin (HE) and Elastica van

Gieson (EvG).

Histochemistry. The cartilage-engineered constructs

were explanted and fixed for cryosectioning by immer-

sion in a 2% formaldehyde solution for 15 minutes. They

were stained for 60 minutes in an Alcian blue solution

(Chroma-Gesellschaft, Koengen, Germany), serially dehy-

drated with ethanol, and then embedded in Eukitt1 (Kindler,

Freiburg, Germany).

Immunohistochemistry. Immunohistochemistry to dete-

rmine type I and type II collagen was done on 7 lm cry-

osections. After fixation in ethanol for 10 minutes, three

washing steps in PBS were performed. For detection of

collagen type II, monoclonal anti-collagen type II anti-

body II-II6B3 was used (1:100 diluted in PBS), which

was provided by the Developmental Studies Hybridoma

Bank (Linsenmayer, IA). For collagen type I detection, a

polyclonal anti-collagen type I antibody (Cell Systems,

Remagen, Germany) (diluted 1:50 in PBS) was used.

RESULTS

Twelve rabbits were operated in this experimental

study; however, one rabbit died intraoperatively due to an

anesthetic overdose and one died postoperatively for

unknown reasons. In total, 10 rabbits were evaluated.

In Vitro Construct Fabrication

After cell isolation and expansion in monolayer cul-

ture, cell number was determined using stain with FDA

(fluorescin-diacetate) and PI (propidium iodine), and

counted using a hemocytometer. Living cells in these

constructs appeared as a green color, whereas dead cells

appeared as a red color (Fig. 2). On average, between

300,000 and 400,000 cells isolated from the biopsies

reached a total of �30 3 106 cells through three pas-

sages. In our study, cells were effectively distributed

throughout the entire construct and adhered well in the

pores of the scaffolds. Nearly 100% vital chondrocytes

were observed within the constructs.

In Vivo Study

Macroscopic observation. All neovascularized carti-

lage-engineered skin flaps in this study were successfully

free-transferred based on the newly implanted vascular

pedicle (Figs. 3A–3D). All free-transferred flaps showed

mild venous congestion in the first postoperative 3 days,

but this spontaneously regressed overtime. Over an obser-

vation period of 2 weeks, these cartilage-engineered skin

flaps showed inconspicuous wound healing with normal

hair growth and color similar to that seen in other

regions. Upon turning over the flap, a newly formed fi-

brous connective tissue capsule could be easily identified

underneath the flap. As a result of this capsule, the

implanted constructs, vascular pedicle, and the skin flap

became integrated together, such that the constructs were

well protected within the skin flap.

Selective microangiography. Selective microangiog-

raphy was supplied for all animals in this study. Microan-

giograms showed a newly neovascularized system in the

cartilage-engineered skin flap established from newly

formed vessels sprouting from the newly implanted vas-

cular pedicle and their innumerable vascular connections

with the originally available vasculature of the flap. All

implanted constructs (both cartilage-engineered and con-

trol constructs) were highly neovascularized with dense

vessel networks around and within the implanted con-

structs (Fig. 4).

Histology. All the neovascularized flaps were vital and

morphologically inconspicuous with normal epidermis,

hair follicles, connective tissue, and blood vessels. Both

implanted cartilage-engineered constructs (Fig. 5) and

control constructs (Fig. 6) were well integrated with the

skin flap, remaining mechanically stable in form and size,

and markedly infiltrated by various cell types and newly

formed vessels into the construct pores. There were no

signs of rejection reaction to the PCL-based polyurethane

scaffolds observed in all neovascularized skin flaps.

Histochemistry and immunohistochemistry. Histo-

chemical Alcian blue stain of cartilage-engineered con-

Figure 2. The result of in vitro cell seeding within scaffold pores

before in vivo implantation using FDA- and PI-stain, 380 (live cells

are green and dead cells are red). [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]



structs within the neovascularized skin flap demonstrated

the presence of hyaline-cartilage-like tissue development

within the constructs. These neocartilage-like tissue

seemed to be homogenous within scaffold pores (Fig. 7)

and not always presented in the entire constructs. Immu-

nohistochemical sections of cartilage-engineered con-

structs were intensively stained for collagen type II.

These neocartilage-like tissue and collagen type II expres-

sion within cartilage-engineered constructs indicated the

ongoing development of cartilage tissue (Fig. 8). Com-

pared with cartilage-engineered constructs, controls with-

out cells showed no signs of neocartilage formation in all

specimens (Fig. 9).

DISCUSSION

Cartilage tissue engineering is becoming a very prom-

ising procedure for clinical cartilage structure repair. To

be successful reconstruction of three-dimensional carti-

lage defects such as the ear, nose, or trachea, suitable

autologous cartilage-engineered constructs must remain

stable in shape and size as well as remain viable on ele-

vation and transplantation.11 Also of first critical impor-

tance is that the structural and mechanical properties of

the scaffolds are optimized. Furthermore, in vitro carti-

lage-engineered constructs must be revascularized and

microsurgically transferred as an axial free flap.

Scaffolds used in cartilage tissue engineering must

fulfill many requirements. They should promote tissue

growth, e.g., cytocompatibility, produce no toxic degrada-

tion products, be able to be sterilized, have suitable pore

size for cell seeding, adhesion, and proliferation as well

as adequate mechanical stability. Biomaterial scaffolds

(e.g., collagen, fibrin, agarose, alginate) can easily result

in adequate three-dimensional cell distribution throughout

the entire construct and allow for the retention of newly

produced extracellular matrix (ECM) within the con-

structs. They have, however, fast degradation rates and

Figure 3. Free microsurgical transplantation of a prefabricated cartilage-engineered flap: (A) flap before free transplantation; (B) the entire

prefabricated cartilage-engineered flap was completely removed based on the newly implanted vascular pedicle; (C) postoperative view

after free flap transfer using microsurgical techniques; (D) the postoperative uneventful wound healing following microvascular transplantation.

[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4. The selective microangiography shows a newly neovas-

cularized vessel system within the skin flap. All constructs were

well neovascularized by dense vessel networks on angiograms.

[Color figure can be viewed in the online issue, which is available

at www.interscience.wiley.com.]

56 Hoang et al.


often lack the mechanical strength required to meet

in vivo requirements. Synthetic materials (e.g., polylactide,

polyglycolide, etc.) can provide the required load-bearing

capacity, appropriate porosity, elasticity, and degradation

time with appropriate properties required for cell adhesion

on the one hand; but on the other hand, they are usually

associated with inadequate cell distribution, increasing cell

dedifferentiation, and poor synthesis of ECM.13,14

In this study, PCL-based polyurethane constructs pur-

chased from PolyMaterial AG, Kaufbeuren, Germany

were used for cartilage tissue engineering. These scaf-

folds are interconnected porous material with pore sizes

�200 lm and porosities about 85%. They consist mainly

of PCL triol and isophorone diisocyanate with high cross-

linking density. The scaffolds have a stiffness comparable

to that of native auricular cartilage with an in vivo degra-

Figure 5. The histology of cartilage-engineered constructs shows a stable integration between constructs and the prefabricated skin flap

(A and B). These porous cell-constructs were well neovascularized by the powerful invasion of newly formed vessels into the construct

pores (HE-stain, 35). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6. The histology of control constructs (without cells) shows histologically marked infiltration of fibrous tissue (HE-stain, 35, A and

B). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7. Alcian blue stain in histochemistry reveals neocartilage-like tissue in cartilage-engineered constructs after in vivo implantation

(310 and 340). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]



dation time of about 1–2 years.13 With respect to the

experimental study of Eyrich et al.13 that cells seeded

directly onto the PCL-based polyurethane scaffolds with

fibrin resulted in a better seeding efficiency and distinctly

more homogenous matrix distribution compared with

without fibrin, we created in this study cell-fibrin-scaffold

constructs for neovascularization and free transplantation.

Our study showed that chondrocytes were effectively dis-

tributed in vitro within the constructs and remained alive

with an ellipse shape within these respective scaffolds.

In vivo, all constructs were embedded and protected well

within the neovascularized skin flap with no signs of any

rejection reaction to the material. The scaffold remained

stable in size and form and exhibited no signs of scaffold

degradation or necrosis. All constructs were well-neovas-

cularized by dense vessel networks on angiograms. Histo-

logically, they were noticeably infiltrated by newly

formed vessels into construct pores. Specific analyses

based on histochemistry and immunohistochemistry dem-

onstrated that the new synthesis of collagen type II as

well as vital cartilage-like tissues were observed in the

cartilage-engineered constructs. In contrast, there was no

evidence of neocartilage tissue development found in the

control group (without cells). Results from our in vitro

and in vivo investigations suggest that PCL-based polyur-

ethane scaffolds offer a suitable material for cartilage tis-

sue engineering.

It is well established that the survival, shape, and

function of all organs in the human body is ensured

based on a defined physiological blood supply. Disruption

of blood perfusion leads to unavoidable organ necrosis.

Correspondingly, in vitro cartilage-engineered constructs,

in which a reliable vasculature is not available, must be

neovascularized by means of providing a reliable blood

supply to ensure their in vivo survival and function.

Most current cartilage-engineered constructs rely on

the application of three-dimensional scaffolds, which will

be absorbed in vivo when the neocartilage structure is

thick or strong enough to support stable in form and

size.11 Our previous studies have showed that in vitro au-

tologous cartilage-engineered constructs could be well

neovascularized, survived, generated, and remained stable

when they are implanted back in vivo subcutaneously or

intramuscularly.10 To provide a reliable blood supply as

well as to free-transfer these in vitro cartilage-engineered

constructs as a combined skin-cartilage-construct-engi-

neered flap corresponding to clinical reconstruction of

combined cartilage and soft tissue defects (such as recon-

struction of entire ear or nose defect), we performed in

this experiment a model of combination between carti-

lage-engineered constructs and skin flap prefabrication

and free microvascular transfer based on the newly

implanted arteriovenous vascular pedicle. Reliable blood

supply for free flap transfer means that the neovasculari-

zation development in this combined skin-cartilage-con-

struct-engineered flap was matured, so that the entire

combined flap was well perfused by the blood flow sup-

plied from the newly implanted vascular pedicle. Reliable

blood supply in the prefabricated flap after 6 weeks of

vascular pedicle implantation was confirmed in this study

Figure 8. Immunohistochemistry of cartilage-engineered construct indicates the development of cartilage-like tissues and collagen type II

expression within constructs (320 and 340). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 9. Immunohistochemistry of control construct showed no

signs of neocartilage formation in all specimens (340). [Color figure

can be viewed in the online issue, which is available at www.

interscience.wiley.com.]

58 Hoang et al.


by selective microangiography, histology as well as by

various experimental investigations in the literature.15–19

Neovascularization of tissue engineering has been

reported on in the literature. In 2001, Tan et al.20

reported in a rat model on vascularizing acellular dermal

matrix (Integra1) with superficial inferiorepigastric ves-

sels and microsurgically transferring them as bioengi-

neered dermal flaps for 72 hours. Tanaka et al.21 wrapped

artificial dermis (1.5 3 2 cm) around a vascular pedicle

and implanted it beneath the rat inguinal skin for 4

weeks. Next, the volume of newly generated tissue within

the artificial dermis was measured or evaluated histologi-

cally. Recently, in 2004 Cronin et al.22 performed in rats

the insertion of a vascular pedicle and matrix material

into a very small closed chamber (0.6 3 0.3 cm), which

was buried subcutaneously. The results in this study

showed that there was evidence of migration into and

survival of native cells within the added matrix, generat-

ing a vascularized three-dimensional construct.

Most studies investigated tissue engineering only

in vitro23,24 or combined in vitro with in vivo implanta-

tion either on nude mice13,25,26 or in autologous animal

models14,27,28 without free microsurgical transplantation.

Moreover, investigation of neovascularization and free

transfer of cartilage-engineered constructs has, to our

knowledge, not appeared in the literature to date. To

allow for extrapolation from experimental investigations

to clinical applications, we implanted in vitro cartilage-

engineered constructs into large prefabricated flaps

(dimension of 8 3 15 cm). In addition, a construct with-

out cultured cells was also simultaneously implanted

within the flap to serve as the control. The experimental

results of this study showed that both cartilage-engineered

constructs and control constructs were macroscopically

well integrated and protected within the prefabricated

flap, which was established as a results of the newly

formed fibrovascular connective capsule underlying the

flap. All neovascularized skin flaps with embedded tissue

engineering constructs and control constructs were suc-

cessfully free-transferred after 6 weeks of prefabrication

using microsurgical techniques. The viability of these

free neovascularized skin flaps was macroscopically con-

firmed by uneventful postoperative wound healing over a

period of 2 weeks.

The effective neovascularization of skin flap as well

as implanted constructs (both cartilage-engineered and

control constructs) in this study was proven on angio-

grams by dense vessel networks within the constructs,

and through histology by good integration between con-

structs and skin flaps as well as a noticeable infiltration

of newly formed vessels into the construct pores. The

histochemistry and immunohistochemistry results showed

that cartilage-like tissue with amount of collagen II syn-

thesis were observed only in the scaffold pores of the

implanted cartilage-engineered constructs. In contrast,

there were no evidences of neocartilage development

detected in the control constructs.

An important question related to this experimental

study is to clarify the role of control constructs, which

were simultaneously implanted with cartilage-engineered

constructs beneath the prefabricated flap. Our experimen-

tal results demonstrated that after in vivo implantation,

porous PCL-based polyurethane constructs in both groups

(TE and control group) were well neovascularized by

newly formed vessels generated from the surrounding

vasculature. In principle, essential differences between

the study group (cartilage-engineered constructs) and the

control group (scaffolds) are that cartilage-engineered

constructs, depending on the newly developed neocarti-

lage cells, should maintain configured three-dimensional

structures stable in form and size when scaffolds were

completely in vivo absorbed. In contrast, three-dimen-

sional structure of scaffolds in the control group will be

totally absorbed and disappeared after 2 years of implan-

tation. Based on this reason, the control constructs used

in this study were served only to determine the existing

of no cartilage cell development within control constructs

following in vivo implantation. In this study, the compar-

ison of neocartilage formation between the study group

and the control group, of our opinion, should be opti-

mized depending on the same animal and the same pre-

fabricated flap.

Concerns regarding construct necrosis due to lack of

the blood supply in using in vitro fabricated tissue engi-

neering constructs were reported by many investiga-

tors.11,29 In this model, the cartilage-engineered construct

was well integrated within the skin flap and successfully

free-transferred based on its axial blood supply pedicle.

In our opinion, these results revealed that the diffusion of

nutrients from the surrounding area in the early phase, as

well as the blood supply from the neovascularization de-

velopment in the next phase, is enough to ensure con-

struct survival, function, and free transplantation.

From a clinical point of view, despite the fact that

cartilage tissue is often required in reconstructive surgery,

the supply of autologous cartilage is limited due to ana-

tomical constraints. In addition, in the clinical practice of

reconstruction of three-dimensional cartilage tissue defect

such as a total ear or nose defect appeared as a result of

burn injuries or tumor resection, there are however situa-

tions in which the appropriate matching local soft tissue

is not available. To optimize functional and esthetic final

results of such reconstructive procedures, prefabrication

of an axial well-vascularized three-dimensional and free-

transferable structures in desired regions by means of

combination between prefabricated flap and cartilage-

engineered configured constructs as a fully done structure

for free microsurgical transfer in the second stage of sur-



gery should be a valuable strategy. On the basis of this

method, we are in interest to hypothesis that appropriate

three-dimensional tissue-engineered constructs of ear,

nose, or trachea required in the clinical routine as well as

other human tissue organs should be performed (Fig. 10).

By using this method, the most important advantage is

that a new three-dimensional cartilage structure with suit-

able shape and form can be generated from a small bi-

opsy by seeding viable expanded cells onto appropriately

configured constructs.1 Subsequently, it is then neovascu-

larized in desired skin regions, regardless of the origin of

the natural vascular anatomy. These neovascularized, car-

tilage-engineered skin flaps can then be safely transferred

as axial free flaps for defect reconstruction. Based on

these advantages of the prefabrication procedure, esthetic

aspects are also markedly improved and donor site mor-

bidity can be significantly reduced.15–19,20–32

However, toward an effective clinical application, fur-

ther experimental investigations have to be underwent

using tissue-engineered constructs of different autologous

tissue such as cartilage, bone, fat, muscle, nerve, etc. to

assess in a relevant implantation. Furthermore, the

in vitro prefabrication of these constructs have to be per-

formed in combination with specific stimulating compo-

nents such as bioreactors, growth factors, serum-free

media, etc., to optimize the success of the procedure.

Nevertheless, the neovascularization and microsurgical

transplantation of in vitro tissue-engineered constructs by

mean of implantation within a prefabricated tissue flap

from this study, in our opinion, appears to be a promising

alternative in the clinical application of cartilage tissue

engineering.

CONCLUSIONS

In conclusion, this study demonstrated the reliable

ability of neovacularization and free microsurgical trans-

plantation of cartilage-engineered constructs using prefab-

ricated flap. In this experimental study, all constructs

6 weeks after implantation were well-protected within the

skin flaps, well neovascularized by blood flow supplied

from the newly implanted vascular pedicle and were suc-

cessfully free-transferred using microsurgical techniques.

Neocartilage development within the implanted cartilage-

engineered constructs was approved by the existence of

cartilage-like tissues and collagen II neosynthesis expres-

sion. With respect to effective clinical application, the

procedure should be a promising alternative for clinical

practice because of favorable esthetic outcomes with min-

imal donor site morbidity.

ACKNOWLEDGMENTS

The authors thank the Alexander von Humboldt Foun-

dation, Germany (AvH-Foundation) for their help in fi-

nancing the author’s research scholarship at the Univer-

sity Hospital ‘‘rechts der Isar’’ in Munich, Germany. The

authors also extend their appreciation to Ms. Christine

Cavanna at the University of Regensburg Medical Center

for her careful editing of this manuscript.

Figure 10. Perspective of clinical applications for reconstruction of three-dimensional cartilage tissue defect (such as an ear defect). [Color

figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

60 Hoang et al.


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Neovascularization and free microsurgical transfer of in vitro cartilage-engineered constructs

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