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HIGHLIGHT

Dendritic Macromers for Hydrogel Formation:Tailored Materials for Ophthalmic, Orthopedic,and Biotech Applications

MARK W. GRINSTAFF1,2

1Department of Biomedical Engineering, Metcalf Center for Science and Engineering,Boston University, Boston, Massachusetts 022152Department of Chemistry, Metcalf Center for Science and Engineering,

Boston University, Boston, Massachusetts 02215

Received 13 October 2007; accepted 25 October 2007DOI: 10.1002/pola.22525Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Dendritic mac-

romolecules are well-defined

highly branched macromolecules

synthesized via a divergent or convergent approach. A salient

feature of the macromolecules

described herein, and a goal of

our research effort, is to prepare

dendritic macromolecules suita-

ble for in vitro and in vivo use

by focusing on biocompatible

building blocks and biodegrad-

able linkages. These dendritic

macromolecules can be subse-

quently crosslinked to form hy-

drogels using a photochemical ac-

rylate-based or a chemical liga-

tion strategy. The properties—

mechanical, swelling, degradation,and so forth—of the hydrogels

can be tuned by altering the com-

position, crosslinking chemistry,

wt %, generation number and so

forth. The utility and diverse appli-

cability is demonstrated through

successful use of these hydrogels

in three unique applications: hy-

drogel adhesives for repairing cor-

neal wounds, hydrogel scaffolds

for cartilage tissue engineering,

and hydrogel reaction chambers

for high throughput screening of

molecular recognition events.

VVC 2007 Wiley Periodicals, Inc. J Polym Sci

Part A: Polym Chem 46: 383–400, 2008

Keywords: adhesives; biologi-

cal applications of polymers; bio-

materials; cartilage; cornea; den-

drimers; dendritic macromole-

cules; high throughput screening;

hydrogels; ophthalmology; ortho-

pedics; structure–property rela-

tionships; synthesis; wound repair

Correspondence to: M. W. Grinstaff (E-mail: [email protected])

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 46, 383–400 (2008)

VVC 2007 Wiley Periodicals, Inc.

383



Mark W. Grinstaff Mark W. Grinstaff is an Associate Professor of

Biomedical Engineering and Chemistry at Boston University. Mark

received his PhD from the University of Illinois under the mentorship

of Professor Kenneth S. Suslick and was an NIH postdoctoral fellow at

the California Institute of Technology with Professor Harry B. Gray.Mark’s awards include the ACS Nobel Laureate Signature Award, NSF

Career Award, Pew Scholar in the Biomedical Sciences, Camille Drey-

fus Teacher-Scholar, and an Alfred P. Sloan Research Fellowship. He

has published more than 90 peer-reviewed manuscripts and given more

than 170 oral presentations. He is a cofounder of two companies that

are commercializing his ideas. His current research activities involve

the synthesis of new macromolecules and amphiphiles, self-assembly

chemistry, tissue engineering, drug delivery, and nanotechnology.

INTRODUCTION

Dendritic macromolecules are finding ever increasing

uses, and in the medical arena are being investigated as

vehicles for drug delivery, contrast agents for imaging,

synthetic vectors for nucleic acid transfection, sealants

for tissue repair, and scaffolds for tissue regeneration

among other uses.1–16 These varied uses are a conse-

quence of the unique compositions, structures, and prop-

erties of these macromolecules. Dendrimers are highly

branched macromolecules possessing three main struc-

tural components: a core, internal branching layers, and

peripheral groups (Fig. 1).17–25 Unlike linear polymers

where growth is accomplished by adding single mono-

mers to the chain (1:1 growth), a dendrimer grows expo-nentially where each monomer is branched leading to

multiple additions (1:2, 1:3, etc. growth). Each layer in a

dendrimer is termed a generation (G) and thus as a den-

drimer grows through the addition of new monomers,

the generation number increases (G0, G1, Á Á Á Gn). As

the generation number of the dendrimer increases, the

structure in solution adopts a globular conformation.

The degree to which a dendrimer attains this globular

shape is determined by the multiplicities of the core and

branches, the orientation of the branching functional-

ities, the flexibility of the branching units, the length of

the repeat unit, and the solvent environment.26–30 Exam-

ples of known peripheral groups included anionic

(CO2À), cationic (NR4

+), neutral (NHC(O)ÀÀCH3), pol-

y(ethylene glycol) (PEG), or alkyl chains and these func-

tionalities play a significant role in the resultant proper-

ties. Taken all together, the chemical and structural

attributes of dendrimers translate to unique chemical and

physical properties (e.g., solubility, chemical reactivity,

viscosity, glass transition temperature).

Dendritic macromolecules are synthesized in a repeti-

tive manner by either a divergent31–38 (from core to pe-

riphery) or convergent25,39–45 (from periphery to core)

approach. As with any synthesis requiring a series of stepwise reactions (e.g., coupling and deprotection reac-

tions), high yields at each step are necessary to ensure

preparation of ample material. Consequently, chemically

well-defined, optimized, and robust reactions are used,

such as amidations, esterifications, hydrogenolysis, and

more recently click chemistry.46 In this highlight article,

I describe (1) the synthesis of the crosslinkable dendritic

macromolecules or macromers; (2) two different chemi-

cal crosslinking strategies with these macromers to pre-

pare hydrophilic macroscopic structures (i.e., hydro-

gels); and (3) the successful application of these hydro-

gels for sealing corneal lacerations and securing corneal

transplants, for repairing cartilage defects, and for creat-ing localized hydrogel reaction chambers for high

throughput screening.

Figure 1. Schematic of an idealized dendrimer.

MARK W. GRINSTAFF

Journal of Polymer Science: Part A: Polymer ChemistryDOI 10.1002/pola

384 J. POLYM. SCI. PART A: POLYM. CHEM.: VOL. 46 (2008)



SYNTHESIS

We are synthesizing and evaluating degradable and bio-

compatible dendritic macromolecules that can be subse-quently crosslinked to form hydrogels. We have reported

the preparation and characterization of a variety of poly-

ester, polyester–ether, and polyamide dendrimers and

dendrons composed of biocompatible building

blocks.13,16,47–57 We call these types of dendritic poly-

mers \biodendrimers." An example of a divergent syn-

thetic approach to a generation fourth poly(glycerol-suc-

cinic acid) ([G4]-PGLSAÀÀOH) dendrimer is shown in

Scheme 1, where a glycerol-succinate monomer is added

to a core using a series of stepwise esterification and

hydrogenolysis reactions.48 Briefly stated, the tetra-func-

tional G0 core, 2, was synthesized in two steps. First, the

monomer, 1 (2-(cis-1,3-O-benzylidene-glycerol)succinic

acid mono ester), was prepared by reacting succinic an-

hydride with cis-1,3-O-benzylidene-glycerol in pyridine.

Next, 1 was coupled to 1.2 equivalents of cis-1,3-O-ben-

zylideneglycerol, in the presence of two equivalents of

N , N -dicyclohexylcarbodiimide (DCC) and 0.5 equiva-

lent of 4-(dimethylamino)pyridinium 4-toluenesulfonate

(DPTS) to yield the core, [G0]-PGLSA-bzld. The blzd

group of the core was removed by hydrogenolysis (10%

Pd/C and H2) in tetrahydrofuran to give the deprotected

core, [G0]-PGLSAÀÀOH, 2. Next, the monomer 1 was

coupled to 2 in the presence of DCC and DPTS followed

by hydrogenolysis to afford the G1 dendrimer. These

esterification and hydrogenolysis reactions wererepeated to give the higher generation dendrimers, and a

G4 PGLSA dendrimer, 5, was prepared.

The convergent synthesis to a generation three ly-

sine–cysteine dendron ([G3]-(Lys)7-Cys8) is shown in

Scheme 2.55 In this example, sequential amide forming

condensation and deprotection reactions were used to

prepare the dendron. The activated pentafluorophenol-

esters of the amino acid building blocks were used,

ZLys(Z)OPFP and IsoCys(Boc)OPFP as this coupling

approach provided the highest yields and cleanest reac-

tions. First, ZLys(Z)OPFP was coupled to LysO-

MeÁ2HCl in the presence of N , N -diisopropylethylamine

(DIEA) and l-hydroxybenzotriazole (HOBT). The Z pro-

tecting group was removed via catalytic hydrogenolysis

(10% Pd/C and H2). For the next step, BocLys(Boc)

OPFP was coupled to the growing dendron. The Boc pro-

tected lysine derivative was used instead of Z protected

since this gave better solubility of the larger Lys-dendron

in organic solvents. Finally, the IsoCys(Boc) OPFP was

added to the dendron and the Boc and Iso protecting

groups of cysteine were removed using trifluoro acetic

acid (TFA) and 1 N HCl in MeOH, respectively, to afford

Scheme 1. Divergent synthesis to a generation fourth poly(glycerol-succinic acid) ([G4]-

PGLSA-OH) dendrimer.


HIGHLIGHT 385



[G3]-(Lys)7-Cys8), 6. In both reaction schemes, the yields

for each individual coupling and deprotection steps were

90% or better. Additional examples of recently prepared

structures besides those shown in Schemes 1 and 2, include

a generation four layered dendrimer composed of succinate

and adapic layers (poly(glycerol-succinic-co-adapic acid)

[G4,G3]-PGLAA-[G2,G1,G0]-PGLSA-OH) 7, a genera-

tion two-one PGLSA dendrimer possessing both carbox-

ylic acid and alkyl chain peripheral groups ([G2-1]-

PGLSAÀÀCO2HÀÀC14) 8, and a generation three hybrid

dendritic-linear macromolecule ([G3]-PGLSAÀÀOH)-

PEG) 9 are shown in Figure 2. Initial cell cytotoxicity stud-

ies show that the dendritic macromolecules possess mini-

mal toxicity and do not induce more death than what is

witnessed in untreated controls.

These synthetic routes to dendritic macromolecules,

whether divergent or convergent, allow for precise com-

positional control within the core, internal branching

layers, and peripheral groups of the macromolecule as

well as the use of a wide-variety of different monomers.

Scheme 2. Convergent synthesis to a generation three lysine-cysteine dendron ([G3]-(Lys)7-

Cys8).

Figure 2. Structures of a generation four layered (poly(glycerol-succinic-co-adapic acid)

[G4,G3]-PGLAA-[G2,G1,G0]-PGLSA-OH) dendrimer, a generation four poly(glycerol-succinic-

acid) dendrimer possessing both carboxylic acid and alkyl chain peripheral groups ([G2-1]-

PGLSA-CO2H-C14), and a generation three hybrid dendritic-linear macromolecule ([G3]-

PGLSA-OH)-PEG).





This level of control during synthesis enables the prepa-

ration of macromolecules possessing a unique molecular

weight or very narrow molecular weight distribution,

unlike most linear polymers. The narrow distribution of

molecular weights allows for the correlation of a specific

physical property, mechanical property, or biological

response to a single specific chemical structure as

opposed to a collection of different molecular weight

structures, as with most linear polymers. The construc-

tion of such structure–property relationships and under-

standings is extremely useful in the design and evalua-

tion of a macromolecule for an intended application.

CROSSLINKING APPROACHES

TO HYDROGELS

Hydrogels are highly hydrated, crosslinked polymeric

networks that are being investigated for a range of bio-

medical applications from drug delivery to scaffolds for

tissue growth.58–62 We have reported two approaches for

preparing hydrogels from these dendritic macromole-

cules. Importantly, both of these methods enable in situ

formation of a hydrogel where an aqueous solution of

the macromer (i.e., the crosslinkable derivative of the

dendritic macromolecule) is delivered to an in vivo site

and is subsequently crosslinked to form a three-dimen-

sional hydrogel that conforms to the shape of the defect.

In the first approach, the hydroxyl peripheral groups of

the dendritic macromolecule are modified, before injec-

tion, to contain a functional group susceptible to free-

radical polymerization, such as an acrylate.13 Upon free-

radical polymerization, many acrylate groups on the

dendritic macromolecules are crosslinked to afford a

hydrogel (Fig. 3). This polymerization reaction can be

initiated using a thermal- or a photo-activated catalyst.

Given our interest in biomedical applications, the need

to work in aqueous solutions, the requirement to work in

the presence of biologics (e.g., proteins and cells), and

the desire to minimize heat generation during the poly-

merization reaction, we have chosen to use a photo-

chemical route. Specifically, we use a visible photoiniti-

ating system that comprises eosin Y, 1-vinyl-pyrrilidi-

none, and triethanol amine. Excitation with an argon ion

laser (kmax¼

514 nm) of an aqueous solution containingthe acrylate-modified dendritic macromolecule and a

small quantity of the eosin Y photoinitiating system ini-

tiates the free radical polymerization of the methacrylate

(MA) moieties on the dendritic polymer. This photoiniti-

ating system has been used for a number of applications

and is nontoxic.61,63–65 The resulting hydrogel is hydro-

philic, transparent, and, depending on the macromer and

solution weight percent, can be soft and flexible or stiff.

In the second approach, the peripheral groups of the

dendritic macromolecule are decorated with nucleo-

philes and subsequently reacted with another polymer

containing electrophiles or vice versa. A number of

nucleophile–electrophile crosslinking chemistries areavailable including the well known reactions of amines

with N -hydroxysuccinimide or thiols with maleimide.

However, we are interested in exploring crosslinking

chemistry which occurs rapidly at 37 8C under neutral

aqueous conditions without the generation of side-prod-

ucts and is amenable to preparing hydrogels with vary-

ing performance lifetimes. Moreover, the reactions must

be chemoselective (i.e., only coupling between the cor-

rect partners) and possess a high tolerance to a range of

other chemical functionalities (e.g., amines, thiols, car-

boxylic acids) that are present under physiological con-

ditions. Consequently, we have selected reactions that

belong to a family of chemical ligations, which havebeen applied successfully to the synthesis of a variety of

proteins.66–70 We are investigating the use of thiazoli-

dine or pseudoproline linkages, which are formed

between an N -terminal cysteine and an aldehyde or an

ester–aldehyde (Fig. 4).55,56 For this approach, the den-

dritic polymer must contain three or more N -terminal

cysteines and the PEG crosslinker must contain at least

two terminal aldehyde groups, or vice versa. Specifi-

cally, we mixed aqueous solutions of a dendron contain-

ing N -terminal cysteines and a PEG-dialdehyde (PEG-

DA) or PEG-diesteraldehyde (PEG-DEA) to afford a

crosslinked network via formation of thiazolidine or

pseudo proline linkages throughout the hydrogel, respec-

tively.55,56 As shown in Figure 4, the amine reacts with

the aldehyde followed by thiazolidine formation, which

is a reversible reaction. If an ester linkage is beta to the

thiozolidine, then an O,N acyl migration occurs afford-

ing the pseudoproline—this step is irreversible. A photo-

graph of one such hydrogel is shown in Figure 5.

Using these two crosslinking strategies, we have pre-

pared a variety of hydrogels for characterization as well

as for evaluation in specific applications. In the follow-

Figure 3. Photochemical crosslinking reaction to form the

hydrogels.


HIGHLIGHT 387



ing sections, three successful applications of the dendri-

tic macromolecules are described. The rationale for the

selection of the specific macromer, the benefits of the

chosen crosslinking approach, and the advantages of our

approach over current methods are highlighted in each

section. Specifically, we will describe the use of (1) pep-

tide-based dendrons and chemical ligation crosslinking

chemistry to repair corneal lacerations and to secure cor-

neal transplants, (2) photocrosslinkable PGLSA-PEG

based dendrimers for cartilage tissue engineering, and

(3) Lys-PEG based dendrimers for creating localizedhydrogel reaction chambers for molecular screening.

OPHTHALMIC APPLICATIONS

The repair of corneal wounds and the restoration of

patient vision are of significant clinical importance. Cor-

neal wounds arise from traumatic injury (e.g., perfora-

tions, lacerations), infections, and surgical procedures

(e.g., transplants, incisions for cataract removal and in-

traocular lens implantation, laser-assisted in situ kerato-

mileusis (LASIK)). Currently, nylon sutures are used to

repair these wounds and depending on the extent of

injury, multiple sutures may be required to secure the

damaged tissue and restore the structural integrity of the

cornea. It is estimated that globally more than 12 million

procedures per year use nylon sutures to close ocular

wounds. However, sutures are not ideal because the

suture solely provides mechanical closure and does not

actively participate in healing, in addition to the suturing

procedure being inherently invasive.71–74 More specifi-

cally, sutures are suboptimal for this application because

(1) the placement of the sutures inflicts additional

trauma to corneal tissue, especially when multiple passes

are needed; (2) sutures can act as a nidus for infection

and incite corneal inflammation and vascularization

increasing the incidence of corneal scarring; (3) corneal

suturing often yields uneven healing, resulting in anastigmatism; (4) sutures are also prone to becoming

loose and/or broken postoperatively and require addi-

tional attention for prompt removal; (5) sutures require

removal by an ophthalmologist, often months after the

operation creating a new opportunity for infection; and

(6) suturing requires an acquired technical skill that can

vary widely from surgeon to surgeon, thus influencing

the overall success of the operation. Consequently, there

is clinical interest in a sealant to replace or supplement

sutures in the repair of corneal wounds.

There are precedents for the use of sealants and these

alternative approaches have had a positive clinical

impact. For example, cyanoacrylate glues were reportedin the 1960s by Webster et al. for the repair of corneal

perforations.75 More recently, fibrin adhesives have been

explored for closing corneal wounds. However, both

these glues have one or more of the following limitations

including ease of application, preparation time, potential

for viral transmittance, heat generation, toxic byprod-

ucts, abrasive materials, and limited effectiveness. A

number of complications have been reported for cyanoa-

crylate glues including cataract formation, corneal infil-

tration, granulomatous keratitis, glaucoma, and retinal

toxicity.76–84 Cyanoacrylate and fibrin glues are used

\off-label" and at the discretion of the surgeon to repair

the wound.Design requirements for an idealized ocular adhesive

generally fall into two main categories. The sealant must

be capable of withstanding a variety of mechanical/opti-

cal constraints present in the ocular environment in addi-

tion to possessing favorable biological characteristics to

Figure 5. Photograph of a hydrogel on top of a package of

nonabsorbable suture.

Figure 4. (Top) chemical ligation reactions yielding either

a thiazolidine (I) or pseudoproline linkage (II). (Bottom)

Dendritic and PEG based macromers for forming the hydro-

gels.





prevent bacterial incursion and to promote native tissue

ingrowth. The ideal material would chemically be capa-

ble of crosslinking on the moist ocular surface in a rapid

and controlled manner, ideally setting in 30 s or less

upon receiving the initiator signal. Additionally, a solu-

tion viscosity (<

100 cP) allows for precise placement of the sealant by the technician. Upon gelation, the result-

ant hydrogel must provide significant closure to main-

tain both the structural integrity of the eye and be capa-

ble of withstanding high intraocular pressures (IOPs)

(>80 mmHg). In addition, the sealant should possess

elasticity greater than that of the corneal tissue to disfa-

vor the formation of an astigmatism. The resultant

hydrogel should also have a refractive index similar to

that of the underlying tissue (1.42) and maintain diffu-

sion properties to allow for gas and nutrient exchange

(>23 10À7 cm2 /s for small molecules). After successful

closure for days to months depending on the extent of

the wound, a characteristic that is tunable with a hydro-gel, the sealant would then be either absorbed or exuded

from the wound.

We have successfully used dendritic macromolecules

as macromers to form hydrogel sealants via the photo-

chemical or chemical ligation crosslinking chemistry to

repair corneal lacerations and perforations,13,16,85,86 seal

cataract incisions,55 secure corneal transplants,56,86 and

close LASIK flaps.85,87 To highlight the importance of

crosslinking chemistry within one type of hydrogel seal-

ant system, we will focus our discussion to full thickness

corneal lacerations and corneal transplants. Corneal lac-

erations that are caused by trauma, infection, inflamma-

tion, or surgical procedures are an ophthalmic emer-gency that can lead to loss of vision. These wounds are

repaired using sutures and as we have discussed earlier,

suturing has significant drawbacks.

Corneal transplantation or penetrating keratoplasty

(PKP) is one of the most common and successful tissue

transplants.88 In a corneal transplantation, the recipient

cornea undergoes a large circular full-thickness cutting

to remove the damaged tissue, after which a previously

cut donor corneal button is manually sutured to the re-

cipient corneal rim. The standard of care today involves

16 running sutures to secure the new transplant tissue in

place. The major disadvantages related to this procedure

include delayed visual recovery, suprachoroidal hemor-

rhage, neovascularization, microbial keratitis, the need

for postoperative suture removal (typically 9 months af-

ter transplantation), and surgically-induced astigma-

tism.89–92

Among the design parameters for these two indica-

tions (corneal lacerations and transplants), the lifetime

of the hydrogel sealant is perhaps the one that necessi-

tates the largest variance in overall requirements. For a

corneal laceration, the sealant must remain in place for

2–4 days to allow for re-endothelization of the corneal

wound site and closure of this relatively small wound

(3–5 mm incision). On the other hand, a sealant for

securing a full-thickness circular 8 mm corneal trans-

plant must perform for months as the host tissue requires

time to integrate with the tissue. To achieve this longev-ity differential, we chose to evaluate the dendron con-

taining N -terminal cysteines ([G2]-(Lys)3-Cys4) and

PEG-DA or PEG-DEA to afford a crosslinked network

via formation of thiazolidine or pseudoproline linkages,

respectively. Hydrogel weight loss, as a function of time

at 25 8C when stored in a humidity chamber, is dramati-

cally different for the two hydrogels. The hydrogel pre-

pared from [G2]-(Lys)3-Cys4 and PEG-DA is intact for

several days whereas the [G2]-(Lys)3-Cys4 PEG-DEA

hydrogel is stable for more than 4 months.56

To determine whether a hydrogel sealant prepared

from [G2]-(Lys)3-Cys4 and PEG-DA would secure a 4.1

mm full thickness corneal laceration, we performed a se-ries of experiments. A 4.1-mm corneal laceration was

made in several enucleated eyes. These wound were ei-

ther left to self-seal, closed using one interrupted 10-0

nylon suture, or closed using the hydrogel sealant. For

the hydrogel sealant, dendron ([G2]-(Lys)3-Cys4) and

PEG-DA were mixed quickly at room temperature and

then approximately 20 lL of the hydrogel sealant was

applied to the wound. A hydrogel was formed upon mix-

ing within 20–30 s as a result of the rapid formation of

thiazolidine linkages. Figure 6 shows a sealed 4.1-mm

corneal laceration repaired using the hydrogel sealant.

Within 5 min of repairing the wound, regardless of the

closure methodology utilized, saline was injected in theanterior chamber via a syringe pump until the repaired

Figure 6. Leaking pressures for hydrogel sealant, sutured,

and untreated 4.1 mm corneal lacerations. Photograph of a

sealed wound (insert).


HIGHLIGHT 389



laceration leaked. In this ex vivo study, the mean leaking

pressure for the hydrogel sealant, sutured, and untreated

eyes (n ¼ 3/sample) were 160, 75, and <10 mmHg,

respectively. The values for the hydrogel sealant and

sutures are above normal IOP of about 12 mmHg. The

wound is not sealed using only the dendron or PEG-DAhydrogel precursors alone but requires the combination

of both to operate effectively. Similar results were

obtained in the treatment of 3-mm cataract incisions.55

The mean leaking pressure for the hydrogel sealant (n ¼

8) and suture (n ¼ 2) treated eyes were 184 and

54 mmHg, respectively. Next, we evaluated if this

hydrogel adhesive would prevent the influx of extraocu-

lar surface fluid into the wound.93 For these experiments,

a cataract incision was made in several additional human

enucleated eyes and then the wounds were either left to

self-seal or treated with the sealant. India ink was

applied to the ocular surface and the IOP was cyclically

raised and lowered between 0 and 100 mmHg six times.Histological analysis showed that India ink entered the

self-sealed wounds but not the sealant-treated corneas.

During the cyclic raising and lowering of IOP, we used

real-time optical coherence tomography to image the ad-

hesive treated wound. Because of its elastic characteris-

tics, the sealant did not dislodge but stretched to con-

form to the wound during the changes in IOP. No leak-

age was observed around the wound site. In regards to

the overall efficacy, the hydrogel sealant secures the cor-

neal wound, provides a water-tight seal, and withstands

higher pressures and stresses placed on a wound than

conventional suture treated wounds. The procedure with

the hydrogel sealant is facile and requires less surgicaltime than conventional suturing, does not inflict addi-

tional tissue trauma, and does not require the use of a

laser—unlike the photocrosslinkable corneal sealants—

which reduces the need for additional instruments as

well as eliminates the small but still present potential

risk from laser eye damage.

With this success, we next determined whether the

hydrogel sealant prepared from dendron ([G2]-(Lys)3-

Cys4) and PEG-DEA would secure the incision between

the host and graft corneal tissue in a transplant. In this

ex vitro model, an 8-mm central corneal trephination

was made in an enucleated eye and then this newly

formed button was autografted back to the original eye.

The host–graft tissue interface was secured using

sutures, sutures combined with the hydrogel sealant, or

the hydrogel sealant alone (Fig. 7). The leaking pressure

for the autografted eyes was measured as we have done

for the corneal laceration studies to determine the extent

to which the wound was sealed.55 The leaking pressure

for autografts receiving 16 interrupted 10-0 nylon

sutures was 13 6 5 mmHg (n ¼ 4). When the hydrogel

sealant was applied (33 wt %; 60 lL) to the sutured

wound in addition to the 16 interrupted sutures, the leak-

ing pressure increased to 63 6 7 mmHg (n ¼ 4).

Increasing the macromer wt % to 50% (60 lL) with 16

interrupted sutures afforded a leaking pressure of 101 6

5 mmHg (Fig. 7). We were unable to secure the auto-

graft to a level above normal IOP when the hydrogel

sealant was used alone indicating that this hydrogel does

not possess sufficient adhesivity by itself to secure a

PKP. However, an additional benefit to this hydrogel

sealant, beyond closing the wound is the potential of thehydrogel barrier formed at the wound interface to pre-

vent the flow of extraocular surface fluid and protect the

wound from postoperative infections. The transport of

India ink across the hydrogel can be monitored as we

have done for the corneal wound study described earlier.

When India ink is applied to the wound, the dye does

not penetrate into the anterior chamber indicating that

the wound interface is secured. The resulting crosslinked

hydrogel sealants are transparent, elastic, hydrophilic,

adhesive, and act as a physical protective barrier to the

ocular surface.

ORTHOPEDIC APPLICATIONS

Osteoarthritis (OA) is a common form of arthritis that

affects 100 million individuals in the world today. In the

early stages of osteoarthritis, proteoglycans and collage-

nous proteins are lost from the cartilage tissue followed

by the formation of small discrete lesions.94,95 As the

disease progresses, these lesions grow and eventually

the subchondral bone is exposed.96–100 This degenera-

tion of articular cartilage leads to a loss of mobility,

Figure 7. Leaking pressures for hydrogel sealant + suture,

suture, and hydrogel sealant treated corneal autografts. Photo-

graph of a sealed hydrogel sealant + 16-sutured corneal auto-

graft (insert).





severe and debilitating pain, and a reduction in the over-

all quality of life for the patients. Depending on the se-

verity of the disease, the current clinical treatments

include the chronic use of anti-inflammatory drugs, abra-

sion, mosaicoplasty, microfracture surgery, and chon-

drocyte transplantation.

94,101–106

The last resort for OApatients is total joint replacement, but this treatment is

costly and traumatic. Yet, these approaches meet with

varied successes due to the lack of a vascular and lym-

phatic system hindering the regenerative capacity of

native cartilage. Cartilage tissue injuries never fully heal

and only worsen with time.94,107 Consequently, there is

significant clinical interest in creating a therapy based

on tissue-engineering principles to restore function to

the damaged cartilage tissue site.

Typically, such strategies to repair cartilage involve a

combination of a polymer-based scaffold, cells, and

growth factors to create the required native carti-

lage.94,108–111

The scaffold plays a key role in the repair of osteochondral defects, and it must meet a number of

design criteria (1) produce a resorbable three-dimen-

sional porous structure in vivo; (2) possess similar me-

chanical properties to the native tissue it is replacing; (3)

support the infiltration, proliferation, and/or differentia-

tion of the required local cell phenotype; (4) be biocom-

patible and nonimmunogenic in vivo; and (5) integrate

with the surrounding matrix in the defect. The scaffold

must ultimately guide the restoration of the tissue during

healing.

Of the various scaffolds materials examined by many

groups, those based on photocrosslinkable hydrogel scaf-

folds are showing considerable promise.59,112–116 Thein situ photocrosslinking ability of these systems is highly

desirable in cartilage tissue-engineering application for a

variety of reasons. First, it allows the uncrosslinked mac-

romer solution to be mixed with cells or soluble factors,

such as growth factors or cytokines, prior to defect site

delivery. Second, the high water content of the scaffold

allows for efficient diffusion of nutrients and oxygen

into, and waste and carbon dioxide out of the hydrogel.

Third, the uncrosslinked macromer solution can easily

flow into irregularly shaped defects common to damaged

or diseased cartilage, facilitating integration with the

surrounding native tissue. Fourth, the liquid state of the

macromer solution allows access to surgically inaccessi-

ble trauma sites via endoscope-assisted (micro)surgery.

Lastly, these materials, once crosslinked in situ, provide

immediate adhesion and mechanical integrity to the

defect site at the time of implantation.

Given our interest in dendritic macromers and hydro-

gels, we evaluated the photocrosslinkable derivatives of

the PGLSA-polyethylene glycol dendritic-linear copoly-

mers (PGLSAÀÀOH)2-PEG as scaffolds for cartilage tis-

sue engineering.65 In addition to satisfying the require-

ments above, these dendritic macromers allow increased

crosslink density of the scaffold without significantly

increasing the polymer concentration when compared

with linear polymer analogs. This approach leads to

improved mechanical properties and minimal swelling

of the hydrogel scaffold, while maintaining (bio)degrad-able sites such as ester linkages throughout the structure.

Specifically, we modified the ([G1]-PGLSAÀÀOH)2-

PEG polymer to contain peripheral terminal MA groups

([G1]-PGLSA-MA)2-PEG (Fig. 8). Once this macromer

is prepared, it can be dissolved in an aqueous solution

containing the visible photoinitiating system (i.e., eosin

Y, 1-vinyl-pyrrilidinone, and triethanol amine) and a

hydrogel is formed upon photolysis with an argon ion

laser at 514 nm. This eosin Y based photocrosslinking

process is mild and has the following benefits: the vivid

pink color of eosin Y in the hydrogel can be easily

observed when placed in the defect site facilitating effi-

cient filling, and the dye is bleached during the cross-linking reaction, confirming reaction completion, and

uniformity of the reaction.

Cylindrical hydrogel samples of known polymer con-

centration and dimensions were prepared and then used

for the swelling, degradation, and mechanical testing in

vitro. Hydrogels of 7.5, 10, and 15 wt % polymer

showed minimal change in shape, gaining in weight only

10% over 30 days in phosphate buffered saline (PBS) at

RT. This is in contrast to linear PEG dimethacrylates

that can swell in excess of 100%. The equilibrium com-

pressive modulus E was dependent on polymer wt %, as

expected, with E increasing significantly from about

3 kPa at the lowest macromer concentration to 600 kPaat the highest macromer concentration (see Fig. 9). The

complex shear modulus |G*| of the hydrogels showed

limited concentration dependence, increasing from about

1–40 kPa, over the concentration range.

Chondrocyte-hydrogel constructs at two different

concentrations (7.5 and 15 wt %) were then prepared

with freshly isolated porcine chondrocytes, placed in

individual wells, and cultured in chondrocyte culture

medium in a humidified atmosphere at 37 8C with 5%

CO2. The chondrocyte-hydrogel constructs were har-

Figure 8. Chemical structure of the ([G1]-PGLSA-MA)2-

PEG macromer used to form the hydrogel scaffolds for chon-

drocyte entrapment.


HIGHLIGHT 391



vested and processed for histology at 4 and 12 weeks

(n ¼ 3). The paraffin-embedded sections were stained

with H and E, Safranin-O (marker for proteoglycans), or

Masson’s Trichrome (marker for collagen) for histologi-cal evaluation. Sections were also immunostained for

the presence of Types I and II collagen. As shown in

Figure 10, the encapsulated chondrocytes showed no

signs of dedifferentiation and retained their rounded

morphology. After 2 and 4 weeks of culture, Safranin-O

and Masson’s Trichrome staining indicated that chon-

drocytes encapsulated in the hydrogels at the lower mac-

romer concentration accumulated significant amounts of

extracellular matrix rich in proteoglycans and collagen,

respectively, (Fig. 10). In contrast, cells encapsulated in

hydrogels at the higher macromer concentration pro-

duced extracellular matrix only in the immediate area

around each cell. Sections of cell-hydrogel constructs

prepared from the 7.5 wt % concentration stained

strongly for Type II collagen demonstrating the accumu-

lation of extracellular matrix with molecular compo-

nents present as found in native articular cartilage. No

significant staining for Type I collagen was observed in-

dicative of fibrocartilage. However, the cell-hydrogelconstructs at 7.5 wt % were degrading over time and, by

4 weeks, some samples had disintegrating into several

smaller fragments. This degradation behavior was not

observed for the cell-hydrogel constructs formed at 15

wt %, even after extended culture time (12 weeks).

Importantly, the 7.5 wt % hydrogel scaffolds were sup-

portive of cartilaginous extracellular matrix synthesis.

However, these hydrogel scaffolds possessed limited

mechanical integrity.

To slow the degradation rate of the hydrogel scaffold

but still retain the favorable characteristics of the 7.5 wt %

hydrogel scaffold in terms of matrix accumulation,

we prepared a new macromer, which contained ester aswell as carbamate linkages.117 We prepared a first gener-

ation dendritic macromolecule composed of glycerol,

succinic acid, b-alanine, and PEG using a divergent

method as shown in Scheme 3. As before, photolysis of

an aqueous solution containing the methacrylated poly

(glycerol beta-alanine)-PEG macromolecule (([G1]-

PGLBA-MA)2-PEG) and the eosin Y photoinitiating

system afforded a crosslinked hydrogel scaffold. The

hydrogel scaffolds at 5, 10, and 20% exhibited no signif-

icant swelling similar to what was seen earlier with the

([G1]-PGLSA-MA)2-PEG based hydrogel scaffolds.

Next, the mechanical properties were measured over a

concentration range of 5–20% w/v. The mechanical

Figure 9. Mechanical properties of the hydrogel scaffold

prepared from ([G1]-PGLSA-MA)2-PEG and ([G1]-PGLBA-

MA)2-PEG compared to articular cartilage.

Figure 10. Histological sections of 7.5 and 15% macromer concentration hydrogels after 2 and

4 weeks incubation. (Left) Red indicates proteoglycans in the Safranin-O stained sections,

(middle) green indicates collagen in the Masson’s Trichrome stained sections and (right) red

indicates Type II collagen in the immunostained sections, no significant Type I collagen was

detected at either concentration. The length of the inserted bar is 100 lm.





properties showed high concentration dependence with

the higher polymer concentrations affording stiffer mate-

rials. Specifically, the mechanical properties of the

hydrogels ranged from about 50–900 kPa for the com-

pressive modulus, and 2–80 kPa for the complex shear

modulus. With respect to native articular cartilage, asshown in Figure 9, the (([G1]-PGLBA-MA)2-PEG)

based hydrogel approaches the mechanical properties of

articular cartilage.

Next, we evaluated the integrity of the hydrogels in a

rabbit knee confined defect model under dynamic me-

chanical testing. Briefly, a simulated osteochondral defect

(3 mm in diameter 3 10 mm in depth) was drilled in the

center of excised medial femoral condyles of New Zea-

land white rabbits and filled with 50 lL of the macromer

and then photo-crosslinked with an argon ion laser for

120 s. The cleaned femur and tibia were then mounted to

the load frame of a custom designed computer controlled,

servomotor-actuator system for simulating rabbit knee ki-

nematics. The hydrogel scaffold was subjected to

dynamic mechanical loading (300 cycles) with a physio-

logically relevant load (30 N at the end of the tibia simu-

lating the body weight of a 3 kg rabbit). Upon completion

of the loading regimen and under visual inspection, the

hydrogel scaffold at 5, 10, and 20% w/v remained intact

in the defect site. The integrity of the hydrogel and the

hydrogel–bone interface was further assessed by mag-

netic resonance imaging. As shown in Figure 11, the 10

wt % hydrogel was still present and integrated with the

surrounding tissue after the dynamic mechanical testing

as were all the other weight percent samples.117

Finally, we conducted an initial in vivo experiment to

evaluate the hydrogel performance in a full-thickness

osteochondral defect. We selected the carbamate–ester– ether (([G1]-PGLBA-MA)2-PEG) hydrogel scaffold

based on its low swelling and high E and G properties.

In addition, the hydrogel scaffold prepared from this

macromer will likely have a longer performance lifetime

than the poly(ester)-based biodendrimers.65 The aqueous

solution containing the crosslinkable biodendrimer and

the photoiniating system was injected into a preformed

defect located in the right knee of adult New Zealand

white rabbits (n ¼ 3). Next the solution was photocros-

slinked using an argon ion laser for several minutes or

until the pink color was gone confirming the crosslink-

ing reaction was completed. A control untreated group

was used in a similar defect located in the left knee of

the same rabbits. At 6 months, the rabbits were sacri-

ficed and histology was performed to determine cellular-

ity (H and E stain), collagen (Masson trichrome), and

proteoglycans (Safranin O stain) content. All three stains

revealed that in the hydrogel scaffold treated defects, the

hydrogel was well integrated with the surrounding tissue

with strong staining for collagen and proteoglycans

(Fig. 12). Importantly, the healing response in the hydro-

gel-filled knees exhibited morphological and biochemi-

Scheme 3. Synthesis of the ([G1]-PGLBA-MA)2-PEG macromer.


HIGHLIGHT 393



cal characteristics consistent with normal–hyaline–tis-

sue, whereas the unfilled controls appeared to be filledirregularly and stained less for collagen and proteogly-

can content. The resulting crosslinked hydrogel scaf-

folds are integrated with the surrounding tissue, mechan-

ically resilient, and promote extracellular matrix produc-

tion in the wound site. These dendritic macromers

possessed a number of favorable properties when used

to prepare scaffolds for the repair of cartilage defects.

BIOTECH APPLICATIONS

Our increasing ability to access and analyze genomic

and proteomic information through microarrays has

afforded substantial scientific and medical advances.

These advances range from greater understanding of

fundamental biological processes to the evaluation of

new drug targets for once untreatable diseases. The ac-

quisition of such biological information relies heavily on

Figure 11. (Left) Photograph of a biodendrimer scaffold filled osteochondral defect. (Right)

Sagittal plane MR image of a biodendrimer scaffold filled osteochondral defect.

Figure 12. Histological section of unfilled (top) and filled (bottom) osteochondral defects. a)

Masson’s trichrome and b) Safranin O staining. Scale bar ¼ 100 lm.





high throughput and high-density screening of molecu-

lar–molecular and molecular–cellular interactions.118–127

Microarray technology is one screening method

which has received considerable attention given that

data can be obtained in a spatially arrayed, high-density

format. However, many of the formats in use rely oncovalent attachment chemistry of one component to a

solid support.123,128–148 This approach affects molecular

and macromolecular properties, requires prior modifica-

tion of the substrate, limits the diversity of assays, and

creates unwanted molecular interactions at the surface—

all of which can influence assay outcome and results.

Eliminating the need for covalent attachment to the sup-

port prior to screening enables investigation of a greater

number and type of molecular interactions while mini-

mizing the biases as a result of the screening approach

undertaken.

Consequently, to address these limitations and de-

velop alternative high throughput screening approaches,we have prepared and evaluated crosslinked immobi-

lized hydrogels as general reaction chambers for screen-

ing (bio)molecular and (bio)macromolecular interac-

tions.149 As described earlier, dendritic macromolecules

possess a number of favorable properties as macromers

for hydrogel formation. For this application, we need to

form small, micron-sized hydrogel reaction chambers on

an aldehyde coated glass surface and thus selected

(Lys)2-PEG in combination with (CHO)2-PEG as the

hydrogel precursors, after a preliminary study of several

potential candidates. The (Lys)2-PEG reacts with the ter-

minal aldehydes of the (CHO)2-PEG as well as the sur-

face immobilized aldehydes to afford Schiff-base link-

ages and a highly crosslinked hydrogel network adhered

to the glass surface. Using an OmniGrid AccentTM

microarraying robot equipped with a Stealth Printhead

containing Stealth Micro Spotting Pins, we dispensed 1

nL volumes of the hydrogel precursors in a solution

containing the (bio)molecule/(bio)macromolecule of in-

terest on the aldehyde modified glass slides (Fig. 13).

After printing, the slides were washed with 1% w/w bo-

vine serum albumin (BSA) in PBS (pH ¼ 7.4) to block

the remaining surface aldehydes in the nonspecific inter-

mediate regions. Importantly, this can all be done under

mild conditions (aqueous solution; pH ¼ 7.4; RT) with-

out the need of prior derivatization of the printed (bio)-

molecule/(bio)macromolecule (Fig. 13). This screening

technique is amenable to high throughput analyses as&50,000 torroid-like hydrogel chambers can be printed

on a single 18 3 72 mm2 glass slide. A photograph of a

reaction chamber is shown in Figure 14.

To evaluate the capability of these hydrogel reaction

chambers for screening small molecule–protein, pro-

Figure 13. Construction of IgG immobilized hydrogels on aldehyde coated slides using

(CHO)2-PEG and (Lys-NH2)2-PEG.

Figure 14. (Left-Top) optical and fluorescent (left-bottom)

image of two hydrogel reaction chambers containing aRNA

(250–5000 nt) (top row) and controls (without aRNA)

(bottom row) after probing with Cy5-aRNA (60–200 nt).

(Right) Example of a screening experiment performed with

an array of reaction chambers.


HIGHLIGHT 395



tein–protein, and nucleic acid–nucleic acid molecular

recognition, we performed a series of well-known model

reactions. For small molecule–protein interactions, we

prepared hydrogel reaction chambers containing biotin

and then probed the chambers with Cy5 labeled strepta-

vidin. The Cy5-streptavidin was successfully incorpo-rated into the hydrogel chambers and bound the biotin.

A twofold increase in red fluorescence relative to control

chamber lacking biotin was observed. Next, we investi-

gated a protein–protein interaction by printing goat IgG

in the hydrogel chambers and then probing with Cy5 la-

beled protein G. A greater than a two-fold increase in

red fluorescence was observed for the hydrogel cham-

bers containing IgG relative to control hydrogel cham-

bers without IgG indicating the formation of the IgG-

protein G complex. A control experiment with BSA

loaded chambers showed no red fluorescence when

probed with the Cy5-Protein G, and, likewise, a reaction

chamber loaded with IgG when probed with Cy5-strep-tavidin showed no red fluorescence. All together, these

data confirm that specific protein–protein recognition

within the scaffold is occurring and that the fluorescence

signals are not merely a result of nonspecific physical

entrapment of the protein in the hydrogel chamber dur-

ing the assay.

The hydrogel reaction chambers are not limited to

studying protein–protein interactions, as we were able to

obtain similar results with nucleic acid–nucleic acid rec-

ognition. As shown in Figure 14, hydrogel chambers

containing fragmented antisense RNA (aRNA) when

probed with complementary Cy5 labeled RNA showed

fluorescence intensities approximately eight-fold greater than controls (unloaded chambers). Probing the RNA

loaded chambers with noncomplementary Cy5 labeled

aRNA confirmed that nucleic acid complementarity was

required and that noncomplementary Cy5 labeled aRNA

was not trapped within the hydrogel chambers. We

extended this work to assessing small DNA strands—a

common screening platform. A 20-mer DNA (50-

TGAGTCTTCTAAGCTCTCCG-30) was printed in the

hydrogel and probed with its Cy5 labeled compliment

(50-Cy5-CGGAGAGCTTAGAAGACTCA-30). After

hybridization, a five-fold increase in red fluorescence

was observed for hydrogel chambers containing the

duplex DNA. Probing the 20-mer with noncomplemen-

tary Cy5-DNA afforded no increase in fluorescence indi-

cating that hybridization had not occurred.

This facile and robotic screening platform using

hydrogel reaction chambers comprised of dendritic mac-

romers offers several advantages over conventional

screening methods and formats. These benefits include

(1) each hydrogel chamber acts as a site-isolated cham-

ber for a specific reaction; (2) the printing and formation

of the chamber occurs simultaneously with the loading

of the (bio)molecule or (bio)macromolecule of interest;

(3) a single platform for all molecular recognition proc-

esses from small molecules to proteins and nucleic

acids; (4) the monitoring of the molecular recognition

events can be achieved in an unbiased facile manner

without modification or chemical attachment of the enti-ties prior to use; and (5) the hydrogel chambers are ame-

nable to the preparation of large arrays for high through-

put screening.

CONCLUSIONS

In summary, dendritic macromers are versatile macro-

molecules for the preparation of hydrogels which are of

interest and utility for a variety of applications. Herein

we described three successful applications using these

macromers and, importantly, each requiring a different

set of design requirements—be it a corneal sealant, ascaffold for cartilage tissue engineering, or a reaction

chamber for screening molecular recognition events. An

underlying theme to this research is the synthesis and

use of dendritic macromolecules that are biodegradable

and biocompatible. For our interests, the syntheses,

whether divergent or convergent, require selection of a

monomer that is known to be biocompatible or degrad-

able in vivo to natural metabolites and high reaction

yields to attain material for subsequent evaluation. Den-

dritic macromolecules are favored in our laboratory over

linear polymers because of the high level of molecular

control that can be achieved during synthesis affording

unique, well-defined macromolecules. This result hastwo significant consequences. First it enables a specific

physical property, mechanical property, or biological

response to be correlated to a well-defined macromolec-

ular composition. Second, it facilitates the designing and

prototyping of a macromolecule for a specific applica-

tion.

With regards to the resulting crosslinked hydrogels

formed from these macromers, there are a number of im-

portant points to learn. First, we can use two different

types of crosslinking chemistries to prepare the hydro-

gels. The photochemical route allows facile \on

demand" crosslinking by application of light and is

adaptable to endoscope-assisted microsurgery. Prior to

crosslinking, the aqueous solution of the macromer can

be applied to the tissue site including those sites that are

difficult to reach or are of irregular size and shape—like

many trauma sites. The electrophile–nucleophile based

crosslinking strategies, which begin to crosslink upon

mixing and then set upon placement on the tissue, do not

require additional instrumentation (such as a laser) for

use but do require careful timing and placement on the

surface. This is true for both the Schiff-base and chemi-





cal ligation chemistry. The chemical ligation strategy is

beneficial since the reaction is performed at neutral pH,

occurs quickly, produces no by-products, and is chemo-

selective. We have investigated both the formation of

thiozolidine and pseudoproline linkages for creating

these hydrogels. These two reactions demonstrate theconcept of using chemical reversibility as a means to

control hydrogel performance lifetimes. The formation

of the thiozolidine is a reversible reaction and thus the

hydrogels prepared using this linkage have limited sta-

bility in water of about a week whereas those hydrogels

prepared with pseudoproline linkages, which involves

an irreversible reaction, remain stable for months. This

difference can be tailored for a specific application as

we have shown for sealing corneal lacerations or secur-

ing corneal transplants.

Using either hydrogel formation strategy, we can vary

the physical and mechanical properties of the resulting

hydrogels. For example, by varying the weight percentor the dendritic structure, the mechanical properties of

the hydrogels can be tuned. We have prepared hydrogels

with a compressive modulus ranging from approxi-

mately 10–900 kPa. Likewise, degradation can be modu-

lated by selecting different linkage chemistries within

the dendrimer structure (e.g., ester vs. amide vs. thiozoli-

dine vs. carbamate). The highly branched structure of

the dendritic macromolecule, which possesses a multi-

tude of crosslinkable groups, allows for efficient cross-

linking and formation of hydrogels with low swelling

characteristics. This is advantageous as excessive swel-

ling can lead to dislodgement of the hydrogel from the

site and/or negate the tissue sealing effect by increasingthe distance between adjacent structures. The hydrogels

can be formed on tissue surfaces and synthetic surfaces

such as glass. In fact, many individual hydrogels can be

prepared on a surface to create arrays for high through-

put screening. Small molecules, proteins, nucleic acids,

and cells can be entrapped within the hydrogels and once

entrapped do not lose their function. Moreover, we have

prepared hydrogels using a wide range of polymer wt %

such that we can form hydrogels that possess from 40 to

93% water by weight. In general, we find hydrogels pos-

sessing such high water weight percents to be biocom-

patible and more suitable for working with biologics

(e.g., tissues, cells, proteins).

These well-defined dendritic macromolecules offer a

wealth of opportunities to control structure and tune

properties. Our studies have enabled a basic understand-

ing of the relationships between composition, structure,

and properties as well as what design requirements are

required for a specific application. It is a chemist’s tool-

box. We can alter composition, crosslinking chemistry,

internal bonds, wt %, adhesivity, generation number and

all of these effects afford diverse macroscopic results.

Continued investigation and development of these den-

dritic macromolecules as well as other biocompatible

compositions and unique architectures will increase our

basic understandings and provide new solutions to

chemical, biological, and medical challenges in the com-

ing decades.

This work was supported in part by the NIH, PEW

Foundation, and BU. The author thanks his collabo-

rators Terry Kim (Duke Eye Center), Brian Snyder

(Children’s Hospital/Harvard Medical School), Lori

Setton (Duke University), Scott Schaus (Boston Uni-

versity) and their fellows and students who worked

on these projects. The author also thanks his gradu-

ate students and postdoctoral fellows for their hard

work and dedication to these projects: Prashant N.

Bansal, Jason Berlin, Michael A. Carnahan, Lovorka

Degoricija, Neel Joshi, Nathanael R. Luman, Steven

R, Meyers, Merredith Morgan, Abigal Oelker, Kim-

berly A. Smeds, Serge H. M. Sontjens, and Michel

Wathier.

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