Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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9.4 Medical Applications

495

spective is that an antibody is made to react

at a site on the protein called an antigenic

determinant or epitope. Several distinct contact

points fixed in space, such as charges and

hydrophobic groups, constitute the epitope. To

fix those points in space in order to constitute

an epitope, an antibody must stop the motions

that are contributing to the favorable free

energy of the elastic protein-based polymer.

This constitutes a barrier to the interaction. In

sum, the energy of stabilization estimated

above for the two bands is 23kcal/mole. Even

if the barrier were one-fourth that sum, for

example, 5.8kcal/mole, the probabihty of inter-

action as required to identify such an epitope

would decrease by a factor of more than

500,000.

Our

hypothesis,

therefore,

is that the remark-

able biocompatibility of elastic protein-based

polymers

arises

from the presence of

mechani-

cal resonances

that

themselves are

result

of the

regular,

nonrandom structure of this family of

entropic

elastic

protein-based polymers.

9A3 A Consilient Approach to

Tissue Engineering

In the words of E.O. Wilson in 1998, "To the

extent that we depend on prosthetic devices

to keep ourselves and the biosphere alive,

we will render everything fragile."^^ The con-

silient approach to tissue engineering utilizes

biology's own materials and mechanisms, con-

cerned with tissue structure and function, to

achieve tissue restoration. It is made possible

by temporary functional scaffoldings composed

of biodegradable and biocompatible elastic

model proteins that are responsive to tissue

variables in the same manner as natural

proteins.

The materials of our approach are natural to

the tissue to be restored; they are protein-based

polymers that are progammably biodegradable;

in their swollen state they degrade to natural

amino acids without release of irritating acid

(as occurs with the commonly used polyglycohc

and polylactic acids); they are elastic and can

match the comphance of the natural

tissue;

they

are biocompatible (the basic sequence in its

contracted state appears to be simply ignored

by the host); and they can have introduced into

them biologically active peptide sequences in a

natural form as part of the designed protein

sequence.

The mechanisms whereby the materials func-

tion are common to the tissue to be restored,

that

is,

they exhibit the same hydrophobic asso-

ciation as occurs in protein structure formation

and function, and the elasticity is due to

damping of internal chain dynamics rather than

due to random chain networks. As protein

function itself is central to cellular function,

this allows elastic protein-based materials in

concert with natural cells to achieve tissue

restoration in a manner entirely coherent with

fundamental relationships between cells and

their natural extracellular matrix.

The materials are fashioned into temporary

functional scaffoldings into which the natural

cells can migrate, attach, spread, and sense the

forces to which the temporary functional

scaf-

folding is subjected, and the cells in response

turn on the genes to produce an extracellular

matrix sufficient to sustain those forces. There-

by the natural cells remodel the temporary

functional scaffolding into a natural tissue.

9.4.3.1 Elastic Protein-based Polymers

Fashioned as Tubes, Sheets, and Fibers

9.4.3.1.1 Elastic Protein-based Polymers

as Tubes

By use of appropriate molds, elastic protein-

based polymers can be shaped into cross-linked

tubes of the desired shape and with a range of

elastic moduli, as shown in Figure 9.18. The

elastic moduH of tube A is about 2 x

10^

Pa, that

of tube B 6 X

10^

Pa, and that of tube C 2 x

10^

Pa, whereas that of the femoral artery is in

the range of 4 to 6 x

10^

Pa.^^'^^

9.4.3.1.2 Elastic Protein-based Polymers

as Sheets

A sheet of 20Mrad y-irradiation cross-linked

(GVGVP)25i, that

is,

X2'-(GVGVP)25i,

shown

in Figure 5.14 and a disk of this material con-

taining a human ureteral explant (see Figure

9.36A, below) is used in the simulated urinary

496

9. Advanced Materials for the Future

FIGURE 9.18. Tubes made by y-irradiation cross-

linking of elastic protein-based polymers of

dif-

ferent compositions. (A) Poly(GVGVP). (B)

Poly[fF(GFGVP), fv(GVGVP)]. (C) Poly(GVGIP).

(Adapted with permission from Urry.^^^ Now to be

credited as www.WorldandIJournal.com)

bladder investigation. A smaller sheet of

chemically synthesized X^^-poly(GVGVP) to

be used in prevention of adhesion studies is

shown in Figure 9.19. These elastic sheets can

also be prepared with a range of elastic moduli.

9.4.3.1.3 Elastic Protein-based Polymers

as Fibers

Chemically cross-Hnked fibers can also be pre-

pared with a range of elastic moduli, break

stresses, and break strains. Two of the more

interesting elastic fibers are shown in Figure

9.20.

The fiber at left exhibits an elastic modulus

almost 10 times greater than that of femoral

artery; it does not break until 350% extension,

and it does so with a stress of one-third of the

20%

elastic modulus. The fiber at right in Figure

9.20 exhibits an elastic modulus a solid 10 times

greater than that of femoral artery: a break

stress of 7

10^

Pa with a break strain of 285%.

Collagen fibers exhibit elastic moduli of about

10^

Pa (two orders of magnitude greater than

that of elastic fibers), but become damaged with

just a few percent extension. On the other hand,

mammalian elastic fibers exhibit elastic moduli

of about 5 X

10^

Pa, but exhibit about 200%

extension. Thus, the chemically cross-linked

FIGURE 9.19. Small sheet of elastic protein-based

material, X^^-poly(GVGVP), to be placed between

the injured, bloodied abdominal wall and the

injured, bloodied, and contaminated loop of bowel

for the prevention of adhesion of bowel to wall.

9.4 Medical Applications

497

100 |xm

Polymer

1 Left Fiber

Right Fiber

E (Pa) at

20%

Strain

3.86

10^

6.12x10^

Break Stress (Pa)

1.26 xlO^

6.94

10^

Break Strain (%)

350

285

FIGURE

9.20. Elastic protein-based fibers a few

hundred micrometers in diameter, prepared by

extrusion and chemical cross-linking. Impressive

mechanical properties of elastic modulus at 20%

extension, of break stress, and of break strain are

listed. (Reproduced with permission from Urry

et al.^0

elastic fibers of Figure 9.20 exhibit elastic

moduli midway between elastic fiber and

collagen but sustain extensions that are about

100 times greater than sustained by collagen.

Formed into meshes or woven or knitted into

tubes and sheets, the elastic fibers in Figure 9.20

would have many uses as prosthetic materials.

When also containing cell attachment se-

quences such constructs could provide tempo-

rary scaffoldings for soft tissue restoration of

many sorts, for example, for vascular wall

restoration, hernial repair, and a host of others.

9.4.4 Soft Tissue Restoration

The family of protein-based polymers empha-

sized here look back to the parent repeating

elastic peptide sequence (GVGVP)n, which was

found in the mammalian elastic fiber, a soft

tissue. Because of this, it is perhaps most ap-

propriate to begin consideration of medical

applications with the subject of soft tissue

restoration. The word restoration describes a

treatment not to replace, as is often implied in

terms of tissue engineering and tissue recon-

struction, but rather to set the stage for the

regeneration of a more nearly natural state.

9.4.4.1 Concept of Designing a Temporary

Functional Scaffolding

Central to the term soft tissue restoration is the

concept of designing temporary, functional

scaffoldings that stimulate the natural cells of

the tissue and, thereby, cause them to regener-

ate the natural functional tissue. The protein-

based polymers of focus here exhibit three

essential enabling properties. First is the capac-

ity to match the elastic behavior and function

of the natural tissue; the second is the capacity

to contain in the model protein sequence cell

attachment sequences, and the third is the

eventual biodegradation of the temporary func-

tional scaffolding, leaving only the natural

regenerated functional tissue in its place.

In natural tissues, cells form multiple attach-

ment sites to their extracellular matrix. By

means of these attachments, cells deform as

the tissue deforms in response to the natural

mechanical stresses and strains that the tissue

must sustain during function. These mechanical

forces constitute the energy inputs that instruct

the cells to produce the extracellular matrix

suf-

ficient to sustain those forces. Thus, an ideal

artificial material should have both the attach-

498

9. Advanced Materials for the Future

ment sites for the natural cells and a compU-

ance that matches the natural tissue.

Elastic protein-based polymers have been

designed both to provide cell attachment sites

and to exhibit the required elastic modulus of

the tissue to be replaced. Thus, this introduces

the potential to design temporary functional

scaffoldings with the capacity to be remodeled,

while functioning, into a natural tissue by the

natural cells of the tissue.

Furthermore, the elastic protein-based mate-

rials themselves have been designed to perform

the set of energy conversions that occur in

living organisms and, in particular, to convert

mechanical energy into chemical signals of the

sort that could provide the stimuH to turn on

the genes for producing the required extracel-

lular matrix.

9.4.4.2 The Key Property of Cellular

Mechano-chemical Transduction

A fundamental property of cells is that they

take instruction from the mechanical forces

to which they are subjected and produce that

extracellular matrix sufficient to sustain those

forces. Evidence for this capacity of cells goes

back more than two decades to the work of

Leung et al.^^ but has more recently been called

cellular tensegrity following more detailed char-

acterization with techniques of modern molec-

ular biology.^^"^"^ An understanding of how this

can occur lies in the design of elastic protein-

based polymers capable of demonstrating

mechano-chemical transduction. The classic

demonstration of a mechanical energy input

resulting in a chemical energy output (i.e.,

mechano-chemical transduction) by elastic

protein-based polymers is found in the experi-

mental result of stretch-induced pKa shifts (i.e.,

pumping protons) in Chapter 5, Figure 5.23.

Figure 9.21 exemplifies the nexus between

cellular mechano-chemical transduction and

elastic protein-based polymers containing cell

attachment sequences that enables these

temporary functional scaffoldings to result in

restoration of natural

tissue.

Figure 9.21 A shows

in the absence of cell attachment sequences that

the elastic matrix X^^-poly(GVGVP) does not

support attachment of

cells.

On inclusion of cell

attachment sequences, as in the chemically syn-

thesized X'^-poly[40(GVGVP),(GRGDSP)],

ligamentum nuchae fibroblasts attach, spread,

and grow to confluence. The micrograph in

Figure 9.21B shows the edge of the plated

area; growth to confluence is complete using

X2^-poly[20(GVGVP),(GRGDSP)], as shown

elsewhere (see Figure 5 of Nicol et

al.^^).

Because of the cell attachments, the effect of

stretching the elastic matrix is to stretch the

cytoskeletal fibers and possibly the integrin

receptors in the membrane, as depicted in the

top and side views of the relaxed (Figure 9.21C)

and stretched (Figure 9.21D) states. Mechani-

cal stretching of the elastic cytoskeletal fibers,

in our concept of mechano-chemical transduc-

tion demonstrated in Figure 5.23, results in

the uptake of protons or, perhaps more to the

point, the release of phosphate (even mechan-

ically driven phosphorylation). Thus chemical

energy output becomes the chemical signal that

directs the nucleus to turn on the appropriate

genes for production of an extracellular matrix

sufficient to sustain the dynamic mechanical

forces sensed in this manner by the cells. The

test of this concept in tissue restoration is

shown below in the simulated urinary bladder

experiment (see Figures 9.36 and 9.37 and

related text, below).

9.4.4.3 Prevention of Postsurgical and

Post-trauma Adhesions

Prevention of adhesions is included within

the application of soft tissue restoration as the

purpose is indeed to restore a soft tissue site to

the normal state that preceded the operation or

other trauma. The several examples discussed

below include abdominal, eye, and spinal soft

tissue sites.

9.4.4.3.1 Abdominal Sites (Emphasis

to Function in a Bloodied,

Contaminated Site)

As shown in Figure 9.22A for the control site,

in this model the abdominal wall is scraped

until it bleeds; a loop of proximal intestine is

punctured until it bleeds and exudes feces; a

loose suture brings the two injured surfaces

into proximity and is tied off in such a manner

9.4 Medical Applications 499

Effect of Stretching on a Cell Attached to an Elastic Substrate

"-poly(GVGVP) B X^"-poly[40(GVGVP),(GRGDSP)]

C Unstretched Matrix

cytoskeletal

integrin «»^«

integrin

Stretched Matrix

stretched

cytoskeletal fibers

Top View

I I Bioelastic Matrices Containing

Cell

Attachment Sequences

e.g. x2"-poly[20(GVGVP),(GRGDSP)]

FIGURE

9.21. Cellular mechanochemical transduc-

tion makes possible tissue restoration by elastic

protein materials containing cell attachment

sequences that function as temporary functional

scaffoldings. The attached cells become stretched as

the matrix is stretched.

The

forces and frequencies of

the stretch/relaxation cycles provide the chemical

signal to the nucleus to turn on the genes for

production of an extracellular matrix sufficient to

sustain the deforming forces.

Thus,

the elastic matrix

made of elastic protein-based polymers provides a

temporary functional scaffolding with the potential

for being remodeled into a natural tissue. See text

for further discussion. (A) X^^-polyCGVGVP). (B)

X2°-poly[40(GVGVP), (GRGDSP)]. (A,B, repro-

duced with permission from Nicol et al.^^) (C)

Unstretched matrix. (D) Stretched matrix. (C,D,

reproduced with permission from Urry.^^^)

Scraped

wall

(bleeding)

Skin

Subcutaneous

tissue -^

Abdominal wall

muscle

peritoneum

Skin

Bioelastic

Matrix

Nylon

stay suture Subcutaneous

tissue ^

Punctured

intestine

(with fecal

'contamination)

Bowel

'OOP Abdominal wall

muscle

peritoneum

Punctured

intestine

(with fecal

contamination)

Nylon

stay suture

Bowel

loop

FIGURE 9.22. Peritoneal cavity model of L. Hoban^^

for the prevention of adhesions following abdominal

wounds: a contaminated, bloodied peritoneal cavity.

(A) The control. An injured, bleeding abdominal

wall forms adhesions to a punctured, bleeding, and

contaminated loop of

bowel.

(B) The test

site.

Adhe-

sions are to be blocked by a sheet of elastic protein-

based material placed between the wall and bowel

and held in place by a loose suture. (Reproduced

with permission from Urry et al.^^)

500

9. Advanced Materials for the Future

FIGURE

9.23. Small sheet of

elastic protein-based material,

X2°-poly(GVGVP), placed

between the injured, bloodied

abdominal wall and the injured,

bloodied, and contaminated loop

of bowel to test the material for

its efficacy in preventing adhe-

sion of bowel to wall. (From

Hoban et al.^^ and Urry et al.^^)

as to be accessible from outside the closed

cavity,

and the abdominal wall

sutured closed.

For the test site, as shown in Figure 9.22B, the

test article, demonstrated in Figure 9.19, is

interposed between bowel and abdominal wall,

as shown in Figure 9.23. One week later the

loose suture is removed, and at 2 weeks the

abdominal cavity is reopened and the site

examined.

In the control, adhesions formed 100% of the

time between abdominal wall and bowel, as

shown in Figure 9.24. At the test site, 80% of

the time there were either no adhesions, as in

Figure 9.25, or insignificant adhesions. Ten

FIGURE

9.24. Control for the prevention of adhe-

sions following abdominal wounds. In the absence of

a sheet of elastic protein-based material, major adhe-

sions form, connecting the loop of bowel to abdom-

inal wall. (From Hoban et al.^^ and Urry et al.^^)

9.4 Medical Applications

501

FIGURE

9.25. Having positioned a small sheet of

elastic protein-based material, as shown in Figures

9.22B and 9.23, between the injured, bloodied

abdominal wall and the injured, bloodied, and cont-

aminated loop of bowel prevented significant adhe-

sion of the bowel to the wall at 80% of the test sites.

(From Hoban et al.^^ and Urry et al.^^)

percent of the time a small slip of adhesion

grew through a tear in the bioelastic sheet or

around the sheet, as shown in Figure 9.26,

where the abdominal wall shows no sign of

inflammation and the sheet has remained trans-

parent without apparent formation of a fibrous

capsule. Another 10% of the time the bioelas-

tic sheet was engulfed by adhesion, but the

mass of adhesion could be opened and the bioe-

lastic sheet simply lifted out. Even within the

mass of the adhesion, there was no attachment

of adhesive mass to the bioelastic sheet.^^'^^

Clearly, in this model 80% of the time 20 Mr ad

y-irradiation

cross-linked poly(GVGVP), X^^-

poly(GVGVP), presented a biocompatible bar-

rier to the formation of adhesions. Under no

circumstances did adhesions form to X^^-

poly(GVGVP),

9.4.4.3.2 Strabismus Surgery (to Prevent

Adhesion Between Rectus Muscle and Sclera

of the Eye)

In the strabismus surgery model in the rabbit

eye,

demonstrated in Figure 9.27, for the

control, the superior rectus muscle is detached

from its point of insertion; a section of muscle

capsule is removed; a similar defect is made in

the sclera of the eyeball, and the rectus muscle

is reattached such that the muscle capsule

defect exactly overhes the scleral defect. One

hundred percent of the time, an extensive adhe-

sion bound muscle to sclera as shown in Figure

9.28. For the test site, as shown in the lower part

of Figure 9.27, a bioelastic sleeve is wrapped

around the rectus muscle. The histological

section in Figure 9.29 shows the bioelastic

sleeve separating sclera (seen as dense con-

nective tissue fibers on the lower right) from

muscle fibers (upper left) to have fragmented

due to sectioning. Importantly, this histologi-

cal section shows no adhesion whatever, and

remarkably there are no inflammatory cells and

no sign of a fibrous capsule as usually forms

around a foreign material.

In this animal model for strabismus surgery/^

20Mrad y-irradiation cross-linked poly

(GVGVP),

X'^-poly(GVGVP), presented a

biocompatible barrier to the formation of

adhesions, caused neither an inflammatory

response nor the usual foreign body response of

fibrous capsule formation, and appeared to be

innocuous, its presence being ignored by the

host.

FKJUKI:

9.26. At \{)% of the lest sites, a small slip oi'

adhesion either grew around (as shown) or through

a break in the sheet of elastie protein-based mater-

ial,

weakly adhering abdominal wall to bowel. Note

at 2 weeks that a librous eapsule does not eover the

sheet and inflammation of the abdominal wall does

not oeeur. (Reprodueed with permission from Urry

et al.^')

Muscle

capsule

defect

Superior

rectus

muscle

Bioeiastic

sleeve

Sclera

FIGURE

9.27. Strabismus surgery model of F. Elsas in the

rabbit eye for the prevention of postsurgical adhesions due

to positioning a bioeiastic sleeve of elastic protein-based

material between rectus muscle capsule and scleral

defects. (Reproduced with permission from Urry et al.^^)

502

9.4 Medical Applications

503

FIGURE

9.28. Control site for the prevention of adhe-

sions in a strabismus surgery model in the rabbit eye

showing, in the absence of a bioelastic sleeve, exten-

sive adhesion between muscle and scleral defects.

(Reproduced with permission from Elsas et al.^^

With permission of Slack Incorporated.)

FIGURE

9.29. Demonstration of the efficacy, in a stra-

bismus surgery model in the rabbit eye created by

Elsas, of a bioelastic sleeve of an elastic protein-

based material to prevent adhesion between scleral

and rectus muscle defects. Note absence of adhesion

and the complete absence of an inflammatory

response. (Reproduced with permission from Elsas

et al.''^ With permission of Slack Incorporated.)

504

9. Advanced Materials for the Future

9.4.4.3.3 Spinal Surgery (Emphasis to

Correct Herniated Intervertebral Discs)

in the Rabbit

The animal model is spinal surgery for laminec-

tomy in the rabbit. Figure 9.30 shows the

exposed spinal surgery (laminectomy) site in

the rabbit model, exhibiting (GVGVP)25i as the

extruded gel and as the difficult to see trans-

parent elastic sheet (membrane). Also shown

are the retractors that hold the site exposed.

From the histology of the control site in Figure

9.31,

a massive fibrous adhesion (EF), is shown

bound to the dura mater (DM) that surrounds

the nerves of the spinal cord (NC) that traverse

from the brain to the tissues. During prepara-

tion of the tissue for histology and the section-

ing process, the dura mater separates from the

spinal cord. The formation of adhesion to dura

is considered by many physicians to be a source

of the continuing pain that follows back surgery

and that results in the failed back surgery

syndrome.^^

When the elastic matrix (EM) made of

X2o_(GVGVP)25i is placed adjacent to the dura,

as shown in Figure 9.32, very little adhesion is

FIGURE 9.30. Spinal surgical (laminectomy) site in

the rabbit model, showing (GVGVP)25i in the gel and

elastic sheet (membrane) states, as well as retractors

(P.

Glazer, surgeon on subcontract from Bioelastics

Research, Ltd.). (Reproduced with permission from

Alkalay et al.''^)