THE ANATOMICAL RECORD PART A 272A:497–502 (2003)

Cell and Organ Printing 2:

Fusion of Cell Aggregates in
Three-Dimensional Gels
THOMAS BOLAND,1* VLADIMIR MIRONOV,2 ANNA GUTOWSKA,3
ELISABETH. A. ROTH,1 AND ROGER R. MARKWALD2
1
Department of Bioengineering, Clemson University, Clemson, South Carolina
2
Department of Cell Biology and Anatomy, Medical University of South Carolina,
Charleston, South Carolina
3
Pacific Northwest National Laboratory, Richland, Washington

ABSTRACT
We recently developed a cell printer (Wilson and Boland, 2003) that
enables us to place cells in positions that mimic their respective positions in
organs. However, this technology was limited to the printing of two-dimen-
sional (2D) tissue constructs. Here we describe the use of thermosensitive
gels to generate sequential layers for cell printing. The ability to drop cells
on previously printed successive layers provides a real opportunity for the
realization of three-dimensional (3D) organ printing. Organ printing will
allow us to print complex 3D organs with computer-controlled, exact placing
of different cell types, by a process that can be completed in several minutes.
To demonstrate the feasibility of this novel technology, we showed that cell
aggregates can be placed in the sequential layers of 3D gels close enough for
fusion to occur. We estimated the optimum minimal thickness of the gel
that can be reproducibly generated by dropping the liquid at room temper-
ature onto a heated substrate. Then we generated cell aggregates with the
corresponding (to the minimal thickness of the gel) size to ensure a direct
contact between printed cell aggregates during sequential printing cycles.
Finally, we demonstrated that these closely-placed cell aggregates could
fuse in two types of thermosensitive 3D gels. Taken together, these data
strongly support the feasibility of the proposed novel organ-printing tech-
nology. Anat Rec Part A 272A:497–502, 2003. © 2003 Wiley-Liss, Inc.

Key words: organ printing; cell printing; cell aggregates; cell

fusion; hydrogel

Several approaches are currently being used to address cated approach. However, gene therapy and cell trans-
the shortage of donor organs, including artificial mechan- plantation, as well as drug therapy, are most effective in
ical organs, xenotransplantation (using animal organs), the early stages of a disease. At terminal stages, or for
tissue engineering, and regenerative medicine. Some ar- injured tissue, there is still a need for organ replacement.
tificial organs are already on the market, but they can Thus it is has been proposed that tissue engineering,
significantly diminish a patient’s quality of life and may
produce unwanted side effects. Xenotransplanation is a
promising approach, especially when it involves the use of
organs from transgenic animals with a reduced capacity to Grant sponsor: NASA EPSCoR Grant; Grant number: HEDS6.
induce acute immune response after transplantation, and *Correspondence to: Thomas Boland, Department of Bioengi-
when it is combined with emerging methods of immuno- neering, Clemson University, Clemson, SC 29634. Fax: (864)
tolerance management. However, there is still great con- 656-4466.
cern about the potential spreading of animal viruses (Coo- Received 28 March 2002; Accepted 17 February 2003
per et al., 2002). Regenerative medicine, or the repair of DOI 10.1002/ar.a.10059
injured or diseased organs in vivo by gene therapy or cell
transplantation, is also a very promising and sophisti-

© 2003 WILEY-LISS, INC.

498 BOLAND ET AL.

which is the growing of organs in vitro from a patient’s

own cells, is a reasonable approach to address the short-
age of donor organs.
The classical tissue engineering approach involves the
use of solid, rigid scaffolds from polyglycolic acid (PGA)
and isolated cells (Langer and Vacanti, 1993). It is based
on the premise that seeding cells in a bioreactor on porous
biodegradable scaffolds will be sufficient to generate or-
gans. However, there are at least four problems with this
method:

1. Cell penetration and seeding is not very effective. It

proceeds on the time scale of months and is not uniform
throughout the scaffold. Although significant progress
has been made in designing scaffolds that enable effec-
tive seeding and cell migration (Ma and Zhang, 2001), Fig. 1. Principle of organ printing. [Color figure can be viewed in the
it is still far from optimal. online issue, which is available at www.interscience.wiley.com.]
2. Organs usually consist of many cell types, and the need
to place different cell types in specific positions is a very
challenging technical problem in solid scaffold design. This technology, which we term “organ printing,” en-
3. The rigid, solid scaffolds made from PLA are not opti- ables the printing of complex 3D organs, with exact plac-
mal for engineering contractile tissue, such as heart ing of different cell types in 3D engineered organs, in only
and vascular tubes. a few minutes. To demonstrate the feasibility of this novel
4. The main problem with using solid scaffold seeding technology, we showed that we can place cell aggregates in
technology (for constructs larger than 200 ␮) is the a 3D gel closely enough for fusion to occur. To do this, we
absence of vascularization. estimated the minimal thickness of gel that can be repro-
ducibly generated by dropping the liquid at room temper-
The lack of vascularization has been addressed by a ature onto heated substrata. Then, on the basis of this
“rolling” approach, which has proven to be very effective experimentally estimated parameter, we designed cell ag-
for building blood vessels (L’Heureux et al., 1998), but it gregates with appropriate sizes to ensure a direct contact
cannot be adapted for complex three-dimensional (3D) between printed cell aggregates during sequential print-
organs. Embedding technology is also very promising (Ne- ing cycles. Finally, we showed that closely-placed cell ag-
rem and Seliktar, 2001), but it can only be applied to gregates could fuse in two types of 3D gels.
multilayer tubular organs, and also suffers from the ab-
sence of a direct placing mechanism. MATERIALS AND METHODS
Recently, rapid prototyping (RP) technology has been Cell Printer Preparation
used to fabricate physical models of hard tissues, tissue
scaffolds, and custom-made tissue implant prostheses A description of the printer is given elsewhere (Wilson
(Potamianos et al., 1998; Holck et al., 1999; Winder et al., and Boland, 2003). Sterile stainless steel needles with luer
1999). RP technology has been used to produce novel scaf- plastic hubs were kept on ice until they were filled with
folds with controllable porosity and channel sizes, poten- the polymer gel of interest. They were then screwed into
tially allowing for vacularization (Zein et al., 2002). Al- the ethanol-sterilized luer fittings of the print head. Care
though the technique is versatile and can reproduce was taken to maintain a needle temperature below 4°C. It
anatomical structures (Sodian et al., 2002), its main lim- is important to maintain a low temperature when working
itation is the lack of polymers with suitable mechanical with collagen because it will gel irreversibly and block the
properties for soft-tissue constructs. (Hutmacher et al., needles at room temperature. The print head and the
2001). This technique also has the drawback that cells printer were placed onto heated petri dishes. The dishes
must be placed in exact positions in the 3D printed scaf- were placed into specially designed aluminum blocks that
fold. were kept at 36°C by a heater (VWR Scientific, Atlanta,
We recently developed a cell printer (Wilson and Boland, GA). The heater, blocks, dishes, and printer were put into
2003) that enables us to place cells in positions that mimic a sterile laminar flow hood.
their positions in an organ. The printer can put up to nine
solutions of cells or polymers into a specific place by the Preparation of Thermosensitive Gels
use of specially designed software, and print two-dimen- A poly[N-isopropylacrylamide-co-2-(N,N-dimethylamino)-
sional (2D) tissue constructs. However, up to now this ethyl acrylate] copolymer, denoted henceforth as K-70,
technology was limited to the printing of only 2D tissue was used for the gel experiments. A molecular weight of
constructs. A new opportunity for extending the printing 510 kD was estimated by gel permeation chromatography
technology to three dimensions is created by the use of (GPC) with a light-scattering detector. The polymer syn-
thermo-reversible gels (Gutowska et al., 2001). Nontoxic, thesis and characterization are described elsewhere (Gu-
biodegradable, thermo-reversible gels, which are fluid at towska et al., 2001).
20°C and gel above 32°C, are used as a sort of “paper” on A 10 wt % polymer solution in cold deionized water was
which tissue structures can be printed, and the cells are put into an external ice bath and stirred magnetically
the “ink.” Successive layers could be generated just by overnight. After the polymer was completely dissolved,
dropping another layer of gel onto an already printed the pH of the solution was adjusted to 7.0 with 0.1 M
surface (see Fig. 1). NaOH, using accurate pH strips. The polymer solution
 CELL AND ORGAN PRINTING 2 499
was sterilized for 30 min in an autoclave, cooled, and then RESULTS
redissolved. The solutions were allowed to become com-
Alternate layers of clear and tryan blue K-70 gel were
pletely clear and were mixed well before further use. The
analyzed under a microscope. Figure 2 shows a top view of
polymer solutions were combined with an equal amount of
the layered gel. The thickness of the layers varied between
2⫻ cell culture medium consisting of Eagle’s minimum
200 and 500 ␮. During the addition of the polymer solu-
medium (MEM) supplemented with 10% fetal bovine se-
tions to the already gelled layers, it was obvious that only
rum (FBS) and 1% antibiotic solution. The final polymer
minimal (if any) mixing of the layers occurred. In the
concentration was not lower than 4 –5 wt %. To visualize
gelled state the polymer is hydrophobic, and one could
the polymer layers, half of the solutions were dyed by
clearly observe that the liquid solution drops did not
adding 30 ␮l of 1% trypan blue dye to each milliliter of
spread out on the gel.
polymer solution.
The size of the aggregates was also measured. We ob-
tained aggregates with an average diameter of 540 ⫾ 183
Preparation of Collagen Gels ␮m (n ⫽ 18). Figure 3 shows a representative image of the
Collagen gels were prepared according to the method of endothelial aggregates used in this study.
Storm and Michalopoulous (1982), with the exception that
lyophilized calf skin collagen rather than rat tail collagen Directed Fusion of Closely-Placed Cell
was used. A 3.33 mg/ml collagen solution was prepared by Aggregates
slowly dissolving the collagen in acetic acid (1%) at 0°C.
The gel was prepared by adding 156.5 ␮l of sterile, filtered To prove that a directed fusion of aggregates could be
10⫻ DMEM and 30 ␮l of sterile 0.34N NaOH to each achieved, we performed an additional series of experi-
milliliter of collagen solution. The resulting solutions were ments using collagen and K-70 gels. Our assumption was
kept on ice until they were used. that directed fusion of closely-placed cell aggregates
should be observed with higher affectivity on the collagen
Estimation of Gel Thickness gel than the thermo-reversible gel. It was shown that
placing cell aggregates in close opposition on the surface of
The thickness of the gel layers was evaluated by depos- collagen type 1 gel resulted in the fusion of adjacent cell
iting a series of drops onto the heated petri dishes. Each aggregates, with a sequential formation of elongated rod-
drop was allowed to gel before a new, smaller drop was like tissue constructs (see Fig. 4). Similar results were
placed on top of the gel. The series of staggered gel layers produced by placing the cell aggregates on the surface of
thus created was placed under a microscope and visual- collagen, and sequentially covering the aggregates with a
ized. The movement (in microns) of the microscope objec- second collagen layer. This proved that fusion of cell ag-
tive was measured as the focal plane shifted from one gregates occurs not only on the surface but also within 3D
layer to the next. collagen gel. Finally, we attempted to reproduce the same
results using the thermo-reversible gel. Although the
Preparation of Cell Aggregates overall effectiveness of the thermo-reversible gels in pro-
Bovine aortal endothelial cells (American Type Culture moting cell aggregate fusion was not equal to that of the
Collection, Manassas, VA) (passage 10) were expanded in collagen type 1 gel, we were able to show that fusion does
T25 culture flasks in the presence of MEM supplemented occur in these gels.
with 10% FBS and 1% antibiotic solution in a 5% CO2 To show that directed fusion can occur on patterned
incubator maintained at 36°C. The media were changed at gels, we loaded the collagen gel into the print head of the
day 1 and subsequently every other day. After growing to printer. The gels were deposited in ring patterns about 1
a confluent monolayer, the cells were washed by replacing cm in diameter and 500 ␮ wide. After gelling occurred, the
the media with isotonic phosphate-buffered saline (PBS). BAEC aggregates were added to the gel. Observation after
They were then incubated in a PBS solution containing a 24 hr revealed that the cells were spread throughout the
millimolar solution of the tripeptide arginine-glycine-as- gel. A live/dead assay image is shown in Figure 5, which
partic acid (RGD) and agitated for 30 – 45 min in the shows that the cells within the gel were alive, whereas
incubator. This caused the cells attached to the edges of most of the cells that migrated out of the gel were not
the flask to dislodge. Further mechanical scraping of the viable.
flask with a sterile glass pipette caused the cells to detach
in aggregates. The aggregates were collected, centrifuged DISCUSSION
at 1,000 rpm for 5 min, and resuspended in 0.5 ml of Directed Cell Placing as the Main Problem of
media. The aggregates were then reseeded on collagen Tissue Engineering
and K-70 gels.
A very important task of tissue engineering is to put
certain cell types in exact places. This cannot be achieved
Live/Dead Assay by traditional bioreactor-based cell-seeding technology us-
The viability of the cells was assessed with a commer- ing porous biodegradable scaffolds (even sophisticated
cially available live/dead assay (Molecular Probes Inc., ones created with prototype printing). Other techniques,
Eugene, OR). The samples were rinsed with PBS, and such as rolling and embedding, are as yet not suitable for
incubated for 30 min in a solution of calcein AM and engineering complex multicellular 3D organs.
ethidium homodimer-1 in PBS according to the manufac- Cell-printing technology using thermo-reversible gel
turer’s protocol. Fluorescence was observed in an inverted provides (at least theoretically) a unique opportunity to
epifluorescent microscope (Nikon Diaphot 300, Nikon Inc., solve this most difficult tissue-engineering task, and
Melville, NY) using a DAPI/FITC/TRITC triple-band fil- opens the way for mass organ printing. This may eventu-
ter. ally lead to a cost-effective solution to the problem of organ
500 BOLAND ET AL.

Fig. 2. Top view of seven alternate layers of clear and tryan blue-
dyed K-70 gel on a collagen-coated dish. The edges between the layers
reveal the amount of mixing that occurs when a cold drop liquifies some
of the gel before it is gelled itself. Optimizing gelling kinetics, drop size,
and deposition rate may minimize this effect. Fig. 4. Cell aggregate fusion on the (A) collagen gel and (B) K-70 gel.

Fig. 3. Image of a single endothelial cell aggregate of ⬃670 ␮m Fig. 5. Cell aggregate fusion on a printed collagen gel ring. The
diameter. image obtained on an epifluorescent microscope shows fused aggre-
gates and cells that migrated across the gel. Cells that migrated toward
the center of the ring (in red) proved nonviable, possibly because they
became dry outside the gel.
shortage. To assess the feasibility of this exciting technol-
ogy, we attempted to remove some of the most obvious
technological barriers to its use. PLA/PGA compositions, microspheres with a range of deg-
radation times could be obtained. Porous microspheres
Estimating the Optimum Minimal Thickness of could be loaded with desired growth factors and dispersed
the Gel Layer within an injectable gel matrix. The release profile of
The presence of the gel layer between the cell aggre- bioactive agents could be adjusted to the required tissue-
gates serves two purposes: 1) it provides mechanical specific spatial/temporal pattern of expression.
strength and stability to the construct, and 2) it serves as
Generation of Sequential Thermo-Reversible
a drug-delivery device. For example, porous PLA/PGA/
PEG polymeric microspheres with controlled degradation Gel Layers
times could be put loaded into the printer for controlled Apart from tissue-specific cell–matrix interactions, the
delivery of growth factors. Using copolymers of different following aspects must be considered: gelation kinetics,
 CELL AND ORGAN PRINTING 2 501
matrix resorption rate, possible toxicity of degradation pared to thermo-reversible gels. This is a more complex
products and their elimination routes, and possible inter- situation than the single-cell or monolayer case described
ference of the gel matrix with histogenesis. above. It is possible that the tethering effect of collagen
There are several possible mechanisms that lead to in mediated by the RGD tripeptide is responsible for keeping
situ gel formation: gelation in response to temperature cell aggregates together, and for their sequential fusion.
change (thermal gelation), pH change, ionic cross-linking, We are exploring ways to optimize the thermo-reversible
or solvent exchange (Guenet, 1992). The kinetics of gela- gel by incorporating RGD or other extracellular matrix
tion are directly affected by the mechanism: thermal ge- analogs (Mann et al., 2001; Griffith and Naughton, 2002)
lation, which is rate-limited by heat transfer, is faster without compromising its thermo-reversible properties.
then ionic cross-linking and pH-triggered gelation, which
are rate-limited by mass transfer. The time it takes for the Toward Organ Printing
gel to form will have an effect on cell spacing and distri- Keeping in mind that the adhesiveness of gels for cells
bution within the printed gel matrix. Gels that form in can be modified, our data show that cell aggregate fusion
response to temperature change may offer specific advan- can be achieved in the thermo-reversible gel. Our data
tages related to fast gelation kinetics. also demonstrate that we can reproducibly generate se-
The thickness of the gel layers therefore depends on the quential layers of thermo-reversible gels of a thickness
kinetics of the gel, as well as on the drop size, which is corresponding to the cell aggregate diameter. This thick-
governed by surface tension and needle diameter. To in- ness allows cell aggregates to be laced in closely enough to
crease the efficiency of the technology, the gel thickness fuse. Both 3D collagen and thermo-reversible gels were
will need to be adjusted to match the size of the aggre- shown to allow fusion of closely-placed cell aggregates.
gates. Using a needle with the same internal diameter as Aggregate fusion causes the 3D structures to shrink some-
the aggregates appears to give satisfactory results. How- what, an effect that will need to be considered in the
ever, as indicated in Figure 2, some distortions in the design of organ blueprints. However, we can also assume
layers (possibly due to a melting process) are observed. that additional extracellular matrix will be formed by the
Further improvements in controlling the aggregate size cells, thus providing additional glue for cell aggregate
distribution would also require a more rigorous model for fusion and serving as a sort of tethering device for pro-
gel thickness. This model would need to take into account moting the self-assembly of printed tissue constructs. Fur-
gelation kinetics, surface tension, and interfacial forces thermore, this additional extracellular matrix may act as
between the liquid and gel states of the solution. a buffer between adjacent structures, and thus be more
forgiving with respect to the exact tolerances of a blue-
Identification of the Optimum Size for Cell print.
Aggregates In addition, the use of gel with RGD ligands may en-
hance adhesion. Alternatively, aggregates trapped inside
The optimum size for cell aggregates is defined as a
gel drops can be printed. Because closely placed drops
diameter that is small enough (usually ⬍1 mm) to allow
fuse, we expect the aggregates to fuse as well. However,
centrally positioned aggregate cells to survive, and a mag-
the time frame of cell attachment is very important, since
nitude that is equal to the thickness of the successively
it determines the speed at which successive layers can be
printed gel layers. The second restraint is much more
printed. Soft-tissue adhesives such as cyanoacrylate es-
important because it determines the overall feasibility of
ters, fibrin sealant, and gelatin-resorcinol-formaldehyde
the technique, whereas apoptosis can be prevented either
glues, or other bioadhesives, could dramatically prevent
by using telomerized cells or cells transfected with blc-2
the constructs from being washed off during successive
(Yang et al., 2001; Nor et al., 2001). Based on estimations
printing cycles. The addition of a corresponding growth
of the optimum minimal thickness, the diameter of the cell
factor in slow-release microspheres could accelerate and
aggregates must be no smaller than 600 ␮m. This does not
direct this process. The programmed release of different
exclude the possibility of variability in cell aggregate size,
growth factors in various concentrations and sequences is
but it definitely places a well-defined limit on the smallest
also technically possible. Finally, the thermo-reversible
technologically acceptable diameter.
gel can be optimized or functionalized by the addition of
extracellular matrix analogs. This would promote cell ad-
Role of Gel and Close Positioning in Promoting
hesion, and result in better cell survival as well as more
the Fusion of Cell Aggregates effective cell aggregate fusion.
The fusion of cell aggregates is a spontaneous phenom- Poor cell survival in the printed aggregates is a possible
enon in aggregate suspension. Recently, important in- drawback to the described technology. We are currently
sights have been gained regarding the possible mecha- exploring different ways to avoid apoptosis and necrosis
nism of aggregate fusion in the presence of extracellular inside the aggregates, such as adding survival factors
matrix (Ryan et al., 2001). It was demonstrated that com- (e.g., basic fibroblast growth factor), using transient ge-
petition exists between cell-to-cell forces vs. cell-to-sub- netic modifications of cells with antiapoptotic (e.g., bcl-2
strate forces. If the cell-to-substrate force is stronger than and telomerase), and blocking apoptotic pathways. It is
the cell-to-cell force, the outcome will be a monolayer. If well known that the survival of cells in cell aggregates can
the force of cell-to-cell contacts is predominant, the cells be seen as a result of cell contact-mediated survival stim-
will form aggregates. This principle was used by us when uli. In this respect, we expect the survival rate of aggre-
we generated cell aggregates. The absence of a cell adhe- gated cells to be higher than that of single printed cells.
sive substrate forced the cells to aggregate in order to Furthermore, this technology can tolerate a small percent-
interact with each other. age of cell death.
Our data demonstrate that collagen gels are more ag- The goals of this study were to demonstrate the feasi-
gregate-friendly and are more “fusogenic” substrates com- bility of organ-printing technology, solve some important
502 BOLAND ET AL.

technical problems, and eliminate some critical technolog- Mann BK, Gobin AS, Tsai AT, Schmedlen RH, West JL. 2001. Smooth
ical barriers. The actual printing of 3D tissue constructs muscle cell growth in photopolymerized hydrogels with cell adhe-
will be the subject of another paper, in which we will also sive and proteolytically degradable domains: synthetic ECM ana-
logs for tissue engineering. Biomaterials 22:3045–3051.
address issues of cell survival, tissue perfusion, and vas-
Nerem RM, Seliktar D. 2001. Vascular tissue engineering. Annu Rev
cularization. Biomed Eng 3:225–243.
Nor JE, Christensen J, Liu J, Peters M, Mooney DJ, Strieter RM,
CONCLUSIONS Polverini PJ. 2001. Up-Regulation of Bcl-2 in microvascular endo-
Taken together, our data strongly indicate that the pro- thelial cells enhances intratumoral angiogenesis and accelerates
posed organ-printing technology is feasible using the orig- tumor growth. Cancer Res 61:2183–2188.
Potamianos P, Amis AA, Forester AJ, McGurk M, Bircher M. 1998.
inally-designed cell printer and thermo-reversible gel.
Rapid prototyping for orthopedic surgery. Proc Inst Mech Eng 212:
383僒393.
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