Fisher John P. e.a. (ed.) Tissue Engineering

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to address focal mucosal disease, underscoring the importance of furthering our understanding of airway

mucosal physiology and pathophysiology.

33.3 Tracheal Tissue Engineering

Tracheal tissue engineering is in its infancy. The ﬁrst true tissue engineering effort appeared only in 1994

[116]. As with any ﬁeld in its early development, the current work is limited by a lack of detailed under-

standing of the physiology and pathophysiology of tracheal tissue. Current work is premised on rather

crude hypotheses. Efforts in this ﬁeld will undoubtedly evolve as functional replacement models begin to

emerge and the critical issues become more apparent. Most efforts have centered on the construction of a

cartilaginous structure that can resist collapse. Recent work has focused on ﬁnding the best cellular source

and method to recreate the cartilaginous infrastructure, and attempting to introduce an epithelial lining

within the lumen of a tissue-engineered construct. Some investigators have made use of mesenchymal

stem cells to create cartilage as well as a respiratory epithelium. Other investigators have explored fetal

surgery techniques allowing their constructs to partly mature in utero. There are also efforts that do not

neatly fall into the category of tissue engineering but are worthy of mention.

Whereas there are many reports of attempted tracheal replacement, it was not until 1994 that the ﬁrst

true tissue-engineering attempt appears in the literature when Vacanti [116] and colleagues reported on

the circumferential replacement of rat tracheas with tissue-engineered cartilaginous tubes. They seeded

chondrocytes harvested from the shoulder of newborn calves onto sheets of nonwoven polyglycolic acid

(PGA) mesh. All experimental animals died within one week of the operation. Presumably, the animals

succumbed to respiratory distress. There was no discussion of postmortem histology; what happened

within the lumen of the airway is not clear. It is likely that a straight cartilaginous tubular structure is

inadequate for two important reasons. First, a rigid tube would place a signiﬁcant mechanical demand on

the anastomoses, encouraging failure at those sites. Second, it would be hard for blood vessels to penetrate

directly through the cartilage to establish the robust vasculature required to meet the high metabolic

demands of airway epithelium. In essence, this design requires that the vascular network arise exclusively

from an intraluminal source and travel from mechanically challenged anastomoses over a considerable

distance, without support from extratracheal vascular ingrowth. Nonetheless, this is a landmark paper in

the evolution of tracheal engineering as it is arguably the ﬁrst true translational effort to tissue engineer

a trachea.

In 2002, Kojima [117] and colleagues addressed the mechanical inadequacies inherent in a straight

cartilaginous tube. They isolated chondrocytes and ﬁbroblasts from the nasal septum of 2-month-old

sheep. Chondrocyte-seeded PGA mesh was placed in the grooves of a 20 by 50-mm-long helical Silastic

template; the entire construct was then wrapped with a ﬁbroblast-laden PGA mesh. In the ﬁrst arm of

the study, the cultured constructs were implanted into dorsal subcutaneous pockets in athymic rats and

explanted after eight weeks. In this reﬁned attempt, the rat-incubated construct yielded cartilage biochem-

ically similar to native tissue as measured by sulfated glycosaminoglycan (GAG) content, hydroxyproline

content, and cellularity. In the second arm of the study, autologous constructs were implanted under-

neath the sternocleidomastoid muscle of the donor sheep, allowed to mature in vivo for eight weeks before

being harvested without preservation of a vascular pedicle and used to replace a surgically created 5-cm

circumferential defect in the cervical trachea of the now four-month-old donor sheep. By contrast with

the results obtained in the rat model, the sheep-incubated constructs were signiﬁcantly more cellular

and poorer in GAG content than native tissue. As a result the sheep-incubated constructs performed

poorly once rotated into the airway, without an attached vascular supply. Postoperatively, none of the

sheep survived for more than seven days. They all suffered from either stenosis or clinically signiﬁcant

tracheomalacia. The difference in the results obtained between the two species was tentatively attributed

to an inﬂammatory reaction engendered by the PGA in sheep, leading to hypercellular cartilage poor

in GAG content, the cartilaginous component thought to be responsible for establishing stiffness. The

authors stated that their future attempts would explore less inﬂammatory scaffolding material and also

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Tracheal Tissue Engineering 33-9

preserve the constructs’ vascular connection to the surrounding muscle. It will be interesting to see if a

helical construct has the appropriate mechanical properties to allow for successful long-term integration

of a tissue-engineered trachea. By its very design, a helix precludes the development of a pars membrana.

Searching for an optimal source of chondrocytes, Fuchs and colleagues [118] experimented in 2002

with the use of fetal chondrocytes to tissue engineer cartilage. Specimens of fetal ovine elastic and hyaline

cartilage were harvested from ears and tracheas, respectively. Adult aural elastic cartilage served as a

comparison. Isolated chondrocytes were seeded onto nonwoven biodegradable scaffolds composed of

poly-l-lactic acid (PLLA)-treated PGA polymer and maintained in vitro for 6 to 8 weeks. The chondro-

cytes isolated from fetal elastic and hyaline cartilage grew signiﬁcantly faster than those harvested from

adult elastic cartilage. All fetal constructs showed characteristics of hyaline cartilage, both on gross and

microscopic inspection, regardless of whether the cells had originated from hyaline or elastic cartilage.

In the fetal constructs, the histologic pattern seen with stains for glycosaminoglycans was comparable to

that seen in native hyaline cartilage. The levels of GAG and collagen type II were higher in fetal versus adult

constructs. There were, however, no signiﬁcant differences between GAG, collagen type II, or elastin in

fetal constructs derived from hyaline or elastic cartilage. When compared to their native tissue, fetal con-

structs derived from elastic cartilage contained similar levels of GAG and type II collagen, but had lower

levels of elastin. Fetal hyaline constructs did not differ signiﬁcantly in the levels of GAG, collagen type II, or

elastin when compared to their native source. Adult elastic constructs, however, were signiﬁcantly lower

in collagen type II and elastin than the native elastic tissue. It is interesting that chondrocytes derived

from elastic cartilage eventually produced hyaline cartilage and grew faster. What governs the formation

of hyaline versus elastic cartilage is unknown at this point. The authors concluded that compared with

adult constructs, matrix deposition is enhanced in engineered fetal cartilage, closely mimicking native

hyaline tissue.

Another study searching for an optimal source of chondrocytes was reported by Kojima and colleagues

in [119] 2003 in which they compared ovine nasal and tracheal chondrocytes seeded onto PGA matrices

and implanted in nude mice. Chondrocytes from the nasal septal cartilage as well as tracheal rings of

2-month old sheep were seeded onto nonwoven mesh of PGA ﬁbers. The cell-constructs were eventually

wrapped around a 7-mm diameter silicone tube and implanted subcutaneously in nude mice for 8 weeks.

They evaluated the gross and histologic appearances, the GAG and the hydroxyproline contents, and the

biomechanical proﬁle of the constructs. The gross appearances from both groups were found to resemble

native tracheal cartilage. The histologic evaluation revealed a mature-appearing hyaline cartilage in both

cases with abundant proteoglycan production. There was no evidence of an inﬂammatory reaction to

the implants. The GAG contents of the tracheal-derived and the nasal septum-derived constructs were

70 and 81% of the native tracheal cartilage respectively. The hydroxyproline contents were 91 and 94% for

the tracheal-derived and the nasal septum-derived constructs respectively. The tensile modulus for both

construct types was, however, only about 15% that of the native trachea. Nonetheless, the constructs were

stiff to the touch and grossly resisted collapse. The authors suggested that other compositional features

of the extracellular matrix, such as collagen crosslinking, might play an important role in the mechanical

proﬁle of tracheal tissue. This study’s ﬁndings support the use of nasal septal cartilage, which is attractive

since it requires a far less invasive procedure for harvest than do most other sources of cartilage.

Later in the same year, Sakata and colleagues [120] reported an attempt to line these cartilaginous tubes

with a respiratory epithelium. They constructed ten tubes as previously described [116] and implanted

them into the ﬂanks of nude mice, this time for six weeks. Epithelial cells were aseptically harvested from

newborn lambs’ cervical tracheas and were immediately injected in the implanted cartilaginous tubes after

which both ends of the cartilaginous tube were sutured shut. The epithelial cell-lined tubes were then col-

lected at intervals of up tothree weeks. Out of ten tubes, six werefound to be infected and had not developed

an epithelial lining as observed on hematoxylin and eosin sections. The remaining four were lined with a

respiratory mucosa, including a submucosal connective tissue, which had “abundant” small blood vessels.

Some of the cells had developed cilia three weeks following inoculation into the implanted cartilaginous

construct. Although promising, this brief report did not provide histologic details. For example the

uniformity of the epithelial lining was not commented upon, and the composition of the epithelium was

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33-10 Tissue Engineering

not reported. The high infection rate highlights one of the challenges of engineering respiratory tissue.

The airway is inherently contaminated with a signiﬁcant bacterial load. Future designs might want to

consider a preliminary in vitro incubation period of the freshly harvested epithelial cells with antibiotics

to reduce the incidence of infection. It might also have been beneﬁcial to attach silastic tubing at either end

of the tubes to allow them to be ﬂushed on a regular interval, as the epithelial lining will begin to produce

mucin and the nonattached dead cells would be trapped within the lumen increasing the risk of infec-

tion. Most importantly, however, this experiment does show that it is possible to line a tissue-engineered

cartilaginous lumen with a respiratory epithelium.

There is ample evidence outside the tissue engineering literature that epithelial cells can be successfully

introduced into a living luminal cavity. Several investigators have made use of a heterotopic tracheal graft

model developed by Terzaghi [121]. These experiments have repeatedly demonstrated that epithelium-

denuded tracheal tissue that is subcutaneously implanted in nude animals can easily be repopulated with

heterologous cultured rat, rabbit, and human epithelial tracheal cells [16,20,121–124]. More recently,

Dupuit and colleagues used human respiratory cells to reconstitute epithelium-denuded rat tracheas,

which were implanted in the ﬂanks of nude mice. Four to ﬁve weeks postimplantation, a fully differenti-

ated and tightly sealed pseudostratiﬁed functional epithelial barrier was documented [125]. Rainer and

colleagues successfully transplanted cultured epithelial cells onto ﬁbrotic capsules surgically created in the

anterior rectus sheath of inbred Wistar Furth rats. They did so by co-culturing native rat tracheal epithelial

cells with irradiated ﬁbroblasts, then suspending them in ﬁbrin glue, which was used as the in vivo delivery

vehicle [126].

A subsequent study by Kojima went one step further by investigating the co-culture of epithelial cells

and chondrocytes. They used ovine nasal septum chondrocytes again, but this time, added nasal septum

respiratory cells suspended in a hydrogel into the lumen of the chondrocyte-seeded constructs in vivo.

The subcutaneously implanted chondrocyte constructs were allowed to mature for six weeks. Histologic

examination, four weeks post instillation of the epithelial cells, revealed that only approximately 60% of

the lumen was covered with an epithelium. Although the epithelial reconstitution was not complete, this

study highlighted the practicality of using a single nasal septal harvest to obtain both cell types [127].

Congenital deformities, such as long-segment stenosis and atresia, in which the tracheal lumen does not

form, are relatively infrequent but bear very high mortality rates. Fuchs and colleagues [128] have begun to

explore the possibility of early in utero intervention using harvested cells seeded onto polymer scaffolding

in an ovine model. The intrauterine environment, bathed in sterile amniotic ﬂuid, offers the advantage of

avoiding the contaminated milieu of the postnatal airway. In their experiments they performed in utero

anterior tracheal replacement using native elastic cartilage (group II) or tissue-engineered constructs

seeded with heterologous fetal chondrocytes (group I).

For group I, fetal elastic or hyaline cartilage was harvested, respectively, from the ear or tracheal

rings. Chondrocyte-seeded PLLA-treated nonwoven PGA mesh was maintained in a rotating oven for

6 to 8 weeks. The constructs were then trimmed into a diamond shape and used to repair a surgically

created longitudinal tracheostomy spanning 4 to 5 cartilaginous rings.

In the second group, fetal auricular cartilage was used as autologous free grafts to repair the same

anterior defect as created in group I. The animals were sacriﬁced prenatally and postnatally. The fetal

and postnatal survival rates ranged from 80 to 100%. Stridor was, however, present in more than half

of the surviving animals. Again, the histologic evaluation of the tissue-engineered grafts revealed hyaline

cartilage formation regardless of whether the cell source was hyaline or elastic cartilage. There was mild to

moderate deformation of the tracheal lumen in some of the tissue-engineered group. In the native cartilage

group, however, the tracheal lumens were severely deformed. The engineered constructs displayed a time-

dependent epithelialization of their lumens. Of note, there were no serous or mucinous acini that are

normally found in native tissue. All the implants of group II retained their elastic cartilage characteristics,

but they also developed a thinner epithelium, which lacked both ciliated cells, as well as serous and

mucinous glands. It is interesting to note that the heterologous chondrocytes engrafted without obvious

signs of rejection. The authors attributed that phenomenon to the immaturity of the fetal immune system

and the low histocompatibility antigen expression of fetal chondrocytes. This study demonstrated the

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Tracheal Tissue Engineering 33-11

feasibility of using heterologous chondrocytes in utero. The authors did not comment on why group II

seemed to have poorer epithelialization; it would be interesting to know if lack of rigidity and possibly

central mobility of the free grafts impaired proper epithelialization and vascularization.

One important note of caution must be mentioned when evaluating anterior tracheal repair studies.

The native trachea has an impressive ability to reform an epithelial mucosa when only a limited amount

of tracheal tissue is removed. This phenomenon is routinely seen after tracheostomy. An epithelial mem-

brane will quickly form after the removal of a tracheostomy tube. Studies that perform only anterior

reconstruction therefore offer a limited understanding of the potential beneﬁts or drawbacks of the graft

materials used.

In 2003, Fuchs and colleagues explored the use of mesenchymal stem cells (MSCs) in tracheal tissue

engineering. MSCs are attractive in tracheal tissue engineering because of their ability to form not only car-

tilage but also muscle with the appropriate biochemical cues. In addition, harvesting MSCs is less invasive

than obtaining autologous cartilage and would obviate the need for an additional general anesthesic and

its attendant risks. MSCs can be harvested from a number of sites. Umbilical cord blood has successfully

been used [129,130]; adipose tissue has yielded a cell population that can undergo chondrogenesis [131].

Though there is at least one report claiming the MSCs can be collected from the peripheral circulation of

an adult [132], traditionally, a bone marrow aspirate is the best means of obtaining a sufﬁcient number

of cells [133].

Fuchs and colleagues showed the feasibility of using MSCs in utero by comparing constructs seeded with

bone marrow-derived heterologous ovine MSCs with constructs seeded with chondrocytes derived from

ovine elastic cartilage. The stem cells were encouraged to differentiate into chondrocytes by exposure to

carefully deﬁned media that contained transforming growth factor as well as ascorbic acid. After 12 weeks

of incubation in a rotating bioreactor, the pretreated PGA mesh-cell constructs were then implanted

in heterologous fashion intoa4to5ringlongitudinal tracheostomy. Normal delivery of the animals

was allowed. Tracheal specimens were harvested 4 days after birth. There were no signiﬁcant differences

found in the survival rates prenatally or postnatally. There was again a signiﬁcant amount of stridor

observed, this time in both groups. The engineered cartilage implants form both sources were found to

engraft despite their heterologous origin; there were, however, some mononuclear inﬁltrates seen in some

specimens. Both groups maintained their cartilaginous phenotype in vivo and both epithelialized their

luminal surfaces. The MSC-derived cartilage had a higher GAG content in vitro, but after remodeling

in vivo, both groups had comparable GAG contents.

In 2004, Kojima explored the use of augmenting an MSC-based construct with transforming growth

factor β2(TGF-β2) released from gelatin microspheres. Bone marrow-derived ovine MSCs were seeded

onto a PGA nonwoven mesh and cultured for a week with exposure to both TGF-β2 and insulin growth

factor 1. The cell-polymer constructs were wrapped around a silicone helical template. Constructs

were then coated with glutaraldehyde-cross-linked gelatin microspheres containing TGF-β2. The entire

construct was subcutaneously implanted in nude rats for six weeks. The proteoglycan content of the

tissue-engineered construct was approximately 79% of that of the native tracheal cartilage. Additionally,

the collagen content of the tissue-engineered product was 71% of that of the native’s content. They found

no statistically signiﬁcant differences in glycosaminoglycan and hydroxyproline content between native

and tissue-engineered tissue. In this instance, the ability to add controlled-release growth factors via

microspheres is of signiﬁcant value as the addition of transforming growth factor has repeatedly been

shown to signiﬁcantly augment chrondogenesis in MSC culture in vitro [134,135].

MSC are attractive not only because of their chondrogenic capacity, but also for their potential ability

to support the respiratory epithelial cells. We recently described a novel in vitro reconstitution system

for tracheal epithelium that could be useful for investigating the cellular and molecular interaction of

epithelial and mesenchymal cells [136]. In this system, a porous Transwell insert was used as a basement

membrane on which MSCs were cultured on the lower side whereas normal human bronchial epithelial

cells were cultured on the opposite upper side (Figure 33.5). When co-cultured with MSCs, respiratory

epithelial cells maintained their capacity to progressively differentiate and form a functional epithelium,

leading to the differentiation of mucin-producing cells.

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33-12 Tissue Engineering

(a)

(c)

(d)

(e)

(f)

(g)

(b)

100 mm

20 mm

0.2 mm

FIGURE 33.5 (a) Schematic representation of the Transwell membrane co-culture of MSCs and respiratory epithelial

cells. (b) The macroscopic observation of the upper side of a Transwell membrane with a characteristic accumulation

of respiratory epithelial cells. (c) Identiﬁcation of MSCs during co-culture on a Transwell membrane with immuno-

staining for vimentin, a mesenchymal cell marker. (d) Identiﬁcation of respiratory epithelial cells during co-culture

on a Transwell membrane with immunostaining for cytokeratin, an epithelial cell marker. (e) Hematoxylin and eosin

parafﬁn section showing a monolayer of MSCs on the bottom, separated from the epithelial cells by a membrane,

which is evidenced by visible pores. (f) Transmission electron microscopy (TEM) revealing the presence of granules

in some epithelial cells. (g) TEM conﬁrming the development of microvilli and actin ﬁlaments the formation of cilia.

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Tracheal Tissue Engineering 33-13

So far most of the work has focused on the infrastructure of tracheal tissue: tracheal cartilage. There

appears to be momentum gathering in this area, and it is likely that a reliable and functional infrastructure

will soon be available. As of today, however, there are no standard endpoints that are commonly accepted

to evaluate the overall functionality and health of the constructs. Presumptively, the ideal properties of

a tissue-engineered trachea are an ability to resist negative and positive pressures, an airtight lining that

promotes a healthy epithelium, torsional ﬂexibility, good vascularization and innervation, and nonim-

munogenicity and biocompatibility to ensure bioincorporation and permanence. As the work matures,

endpoints are likely to routinely include thorough histological evaluation of the airway epithelium,

vascular and neural networks, as well evaluation of biomechanical and biochemical function.

A major challenge in tracheal tissue engineering is the avoidance of the mucosal inﬂammatory response

that leads to its cicatrization and an eventual reduction and obstruction of the lumen. Though recent work

has begun to elucidate some of the details, the inﬂammatory cascade involved in this phenomenon is far

from being well understood. The use of microchip arrays may help to shed some light on some of these

biochemical events and guide our eventual designs. The ultimate success of a tissue-engineered trachea is

likely to be dependent on future basic science efforts in airway mucosal physiology.

It is likely that the promotion of thorough vascularization and innervation will be integral to the health

of the construct. So far, very little work has been done to evaluate vascularization. It is presumed, but not

known, that complete coverage with a healthy respiratory epithelium at the time of airway implantation

will reduce, if not eliminate, the inﬂammation so often seen. Considering the high metabolic nature of

epithelial cells, an adequate network of blood vessels is likely to be a prerequisite. It is feasible that as our

understanding of mucosal physiology matures, we might potentially avoid a two-stage operation, but, in

the short term, this is unlikely to be the case. Little or no work has focused on the innervation of the tissue-

engineered constructs. Though poorly understood, it is likely that neural input is integral to the healthy

maintenance of airway epithelium [6]. How much time will be needed to establish robust circulation

and innervation? Will growth factors be necessary? Will the development of an airway bioreactor be

advantageous?

At this point, it is not known if a respiratory epithelium is an absolute must; at the very least, the surface

must be airtight to prevent bacterial colonization. The presumption is that a respiratory epithelium

is the ultimate lining, but could an interim lining also work until the native cells reline the lumen

[137]? Respiratory epithelial restitution takes place rapidly in native tissue. Would a respiratory epithelial

cell-friendly surface, such as a basement membrane, sufﬁce? Over what length could we expect the

native epithelial cells to migrate? Will tissue-engineered epithelium be functional and display coordinated

movement of its cilia?

Tracheal tissue engineering is currently in the very early stages of its development. As with any new

effort, there is great hope that it will produce signiﬁcant advances in our ability to treat tracheal disease.

Its potential, however, is inescapably restrained by our limited understanding of airway physiology and

will only ﬂourish with persistently diligent efforts in both biomaterials engineering and basic science.

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