Acta Biomaterialia 1 (2005) 377–385

ActaBIOMATERIALIA

www.actamat-journals.com

Regulation of cellular infiltration into tissue engineeringscaffolds composed of submicron diameter fibrils

produced by electrospinning

T.A. Telemeco a,1, C. Ayres b,2, G.L. Bowlin b,3, G.E. Wnek c,4, E.D. Boland b,5,N. Cohen d,6, C.M. Baumgarten e,7, J. Mathews b,8, D.G. Simpson f,*

a Shenandoah University Division of Physical Therapy, Winchester, VA 22601, United Statesb Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA 23298, United States

c Department of Chemical Engineering, Case Western Reserve University, Cleveland, OH 44106, United Statesd Medical College of Virginia Hospitals, Virginia Commonwealth University, Richmond, VA 23284, United States

e Department of Physiology, Virginia Commonwealth University, Richmond, VA 23298, United Statesf Department of Anatomy and Neurobiology, Virginia Commonwealth University, 1101 East Marshall Street,

Richmond, VA 23298, United States

Received 26 January 2005; received in revised form 4 April 2005; accepted 6 April 2005

Abstract

We characterize the infiltration of interstitial cells into tissue engineering scaffolds prepared with electrospun collagen, electro-

spun gelatin, electrospun poly(glycolic) acid (PGA), electrospun poly(lactic) acid (PLA), and an electrospun PGA/PLA co-polymer.

Electrospinning conditions were optimized to produce non-woven tissue engineering scaffolds composed of individual fibrils less

than 1000 nm in diameter. Each of these materials was then electrospun into a cylindrical construct with a 2 mm inside diameter

with a wall thickness of 200–250 lm. Electrospun scaffolds of collagen were rapidly, and densely, infiltrated by interstitial and endo-

thelial cells when implanted into the interstitial space of the rat vastus lateralis muscle. Functional blood vessels were evident within

7 days. In contrast, implants composed of electrospun gelatin or the bio-resorbable synthetic polymers were not infiltrated to any

great extent and induced fibrosis. Our data suggests that topographical features, unique to the electrospun collagen fibril, promote

cell migration and capillary formation.

� 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Electrospinning; Electrospun collagen; Electrospun PGA; Electrospun PLA; Tissue engineering

1742-7061/$ - see front matter � 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.actbio.2005.04.006

* Corresponding author. Tel.: +1 804 827 4099; fax: +1 804 828 9477.

E-mail addresses: ttelemec@su.edu (T.A. Telemeco), ayresce@vcu.edu (C. Ayres), glbowlin@vcu.edu (G.L. Bowlin), gewnek@mail1.vcu.edu

(G.E. Wnek), edboland@mail2.vcu.edu (E.D. Boland), nmcohen@mail2.vcu.edu (N. Cohen), baumgart@mail2.vcu.edu (C.M. Baumgarten),

jmathew@mail2.edu (J. Mathews), dgsimpso@vcu.edu (D.G. Simpson).1 Tel.: +1 540 545 7398; fax: +1 540 665 5530.2 Tel.: +1 804 827 4099; fax: +1 804 828 9477.3 Tel.: +1 804 828 2592; fax: +1 804 827 0290.4 Tel.: +1 804 828 7790; fax: +1 804 828 3846.5 Tel.: +1 804 828 2594; fax: +1 804 828 9477.6 Tel.: +1 804 828 2755; fax: +1 804 827 1016.7 Tel.: +1 804 828 4773; fax: +1 804 828 7382.8 Tel.: +1 804 828 2592; fax: +1 804 827 0290.

378 T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385

1. Introduction

The reconstruction of dysfunctional organs with bio-

engineered tissue holds enormous promise [1]. However,

to fulfill this promise, two broad criteria must be met.

First, reliable, and suitable, sources of donor materialmust be identified. Steady advances in stem cell biology

and nuclear transplantation are likely to identify donor

material for a variety of tissue types in the near term.

Second, as donor technology continues to evolve, it is

becoming increasingly clear that tissue engineering scaf-

folds will play a critical role in the bioengineering para-

digm [2]. Considerable effort has been directed at

developing proteins of the collagen family into tissueengineering scaffolds [3,4]. As a family, the collagens

are highly conserved, represent the principal structural

elements of the interstitium and are the most abundant

protein species of the mammalian system. However,

the methods commonly used to isolate and reprocess

proteins of the collagen family into tissue engineering

scaffolds compromise many of the structural and func-

tional properties of this natural polymer. Type I colla-gen can be isolated from dermis or tendon by acid

extraction. When this acid soluble fraction is returned

to physiological ionic concentration and neutral pH,

the collagen forms a hydrated gel [5]. The poor struc-

tural integrity and labile nature of the collagen gel limit

the usefulness of this type of isolate as a tissue engineer-

ing scaffold. Synthetic [6] and hybrids of synthetic and

natural materials [7] have been used in efforts to circum-vent the structural limitations that are inherent to the

collagen gel. Unfortunately, the synthetic components

of a hybrid material can induce inflammation [6,8], a

foreign body reaction and fibrosis, even when the cellu-

lar components of the bioengineered construct escape

immune surveillance [9].

In this study we use the process of electrospinning to

fabricate tissue engineering scaffoldings from a varietyof different materials. Electrospinning uses an electric

field to process polymers into discreet fibrils. In this pro-

cess, a polymer solution or melt is charged by a high

voltage and directed towards a grounded target [10].

The electric potential drives, or pulls, the polymer solu-

tion across an air gap and the solvent carrier evaporates.

Depending upon the reaction conditions electrospinning

can be used to produce a fine aerosol of particles or anon-woven matrix composed of sub-micron diameter fi-

brils. This size scale is far smaller than can be achieved

with conventional processing methods and approaches

the diameter of collagen fibrils present within the native

extracellular matrix [11]. While electrospinning technol-

ogy can be used to fabricate sub-micron diameter fibrils

on a large scale, it is unclear if this type of engineered

matrix can support cell migration, a process that is crit-ical to the integration of implanted materials into the

host.

In our experiments, conditions were optimized to

deposit the biocompatible/biodegradable polymers

poly(glycolic) acid (PGA), poly(lactic) acid (PLA), the

natural polymer type I collagen (calfskin), and type I gel-

atin, an isolate of collagen that has been denatured prior

to electrospinning, into scaffoldings composed of indi-vidual fibrils ranging from 500 nm to 1000 nm in diame-

ter. Electrospinning under the conditions used in this

study produces a dense matrix composed of randomly

arrayed fibers. Our objectives are to examine how inter-

stitial cells interact with tissue engineering scaffolds

composed of relatively small pore dimensions and sub-

micron diameter fibers. We discuss how the composition

of these fibers modulates cellular infiltration, an eventthat is critical to the process of integrating implanted

materials into the surrounding tissue of the host.

2. Materials and methods

2.1. Electrospinning

Reagents were purchased from Sigma Aldrich (St.

Louis, MO) unless noted. Type I collagen was prepared

by acid extraction. Calfskin corium was cut into 1 mm2

blocks and incubated for 24 h at 4 �C in acetic acid (0.5

M) under constant stirring. All subsequent processing

of collagen was conducted at this temperature. The ex-

tracted calfskin was filtered through cheese cloth and

centrifuged at 20,000G for 12 h. The supernatant wasrecovered; the acid soluble fraction was supplemented

to 3.5 M NaCl and re-centrifuged at 20,000G for 12 h.

Pellets were recovered, re-suspended in 0.5 M acetic acid

and then dialyzed against ice cold, ultrapure 18 Meg-

Ohm water. Final collagen isolates were frozen and

lyophilized to a dry powder. Collagen prepared under

these conditions is subsequently referred to as ‘‘acid sol-

uble type I collagen.’’PGA [12], PLA [13], PGA/PLA co-polymer (Alker-

mes, Cambridge, MA), lyophilized calfskin type I gelatin

[14] and lyophilized acid soluble calfskin type I collagen

[11] were each suspended for 12 h at varying concentra-

tions (60–140 mg/ml) in 1,1,1,3,3,3-hexafluoro-2-propa-

nol (HFIP) for electrospinning [15]. Polymer solutions

were placed into a syringe capped with a blunt tipped

needle and charged to 20 kV. The source solution wasseparated from the ground target by an air gap of

approximately 125 mm. A rotating, 2 mm diameter cylin-

drical mandrel was used as a ground target. Each mate-

rial was electrospun to form a cylindrical construct with

a final wall thickness of 200–250 lm and an overall

length of 20–25 mm (Fig. 1). Electrospinning conditions

were optimized in preliminary experiments to produce

constructs composed of fibers ranging from 500 to1000 nm in diameter. Fiber diameter was modulated by

regulating the starting concentrations of each polymer

Fig. 1. Surgical placement of electrospun constructs (A). For biocom-

patibility testing each material was electrospun into a cylinder 20–25

mm in length with a 2 mm I.D. and a wall thickness of 200–250 lm.

The distal ends of electrospun cylinders were sutured shut with 50 silk

to form a hollow enclosed space (inset). The arrows in panel A denote

the surface of a cylindrical construct placed within a blunt dissected

channel in the rat vastus lateralis muscle immediately prior to wound

closure.

T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385 379

in the initial electrospinning solutions. In this study type

I calfskin collagen was spun at 80 mg/ml HFIP (averagefiber diameter 700 nm), type I calfskin gelatin was spun

at 100 mg/ml HFIP (average fiber diameter 600 nm),

PGA, PLA and the PGA/PLA co-polymer were each

spun at 110 mg/ml HFIP (average fiber diameter for each

of the synthetic polymers was 800 nm). All constructs

were placed into sealed 100 ml dishes and cross-linked

in 250 ll of 50% glutaraldehyde vapor for 1 h at room

temperature. The fixation of the collagen and gelatin-based materials is necessary to stabilize the structure of

the electrospun fibrils. If fibrils of electrospun collagen

or electrospun gelatin are immersed in an aqueous sol-

vent without fixation these materials will nearly instantly

dissolve. Fibrils of electrospun PGA, PLA and the PGA/

PLA co-polymer do not require fixation. However, in an

attempt to maintain uniform experimental conditions we

elected to fix all of the electrospun samples using thesame protocol. At the conclusion of the fixation interval

all samples were rinsed in 70% alcohol and transferred

immediately to a solution of 0.1 M glycine prepared in

ultrapure water. The glycine incubation step was done

to block any residual aldehydes that might be present

after the fixation process. Constructs were then rinsed

three times in ultrapure water and incubated in sterile

PBS for 15 min prior to surgical placement.

2.2. Scanning electron microscopy

Relative fibril size and average pore dimension were

determined from vapor fixed samples that were pro-

cessed for conventional scanning electron microscopy

(SEM). SEM images were digitized and analyzed with

NIH Imagetool. All measurements were calibrated from

size bars incorporated into the SEM images at the time

of capture. Average fibril diameter was determined from

measurements taken perpendicular to the long axis ofthe fibrils within representative microscopic fields

(n = 8 for each sample, 50 fibers per field were mea-

sured). Pore areas (a measure of porosity) were deter-

mined by measuring the area encompassed by adjacent

fibrils in the SEM images (n = 8 for each condition with

20 measurements taken per field).

2.3. Gel analysis

Samples of unfixed electrospun collagen and electro-

spun gelatin were placed into distilled water at a con-

centration of 2 mg/ml and allowed to dissolve at room

temperature. The samples were then diluted 1:1 with

SDSloadingbuffer (62.5mmol/lTris–HCl,10.0%glycerol,

and 4.0% SDS; pH 6.8), final protein concentration 1 mg/

ml. Proteins were separated by conventional SDS gel elec-trophoresis under non-reducing conditions on a 7.5% gel.

GelswerestainedwithCoomassiebrilliantblue,de-stained

and photographed after published methods [16].

2.4. Implant studies

Implant studies were conducted to characterize the

relative performance of electrospun PGA, PLA, PGA/PLA co-polymer, calfskin type I collagen and gelatin

prepared from calfskin at supporting cell infiltration.

Electrospun cylindrical constructs were prepared, im-

planted into the belly of the vastus lateralis of the rat

(Fig. 1 and inset) and recovered after seven days for

microscopic evaluation. These experiments assume that

scaffold formulations that support cellular migration/

infiltration will accumulate interstitial and endothelialcells from the surrounding tissue of the host. Under

these circumstances the electrospun matrix should be

infiltrated and the central lumen of the construct should

be populated by a variety of cell types. Conversely, if a

given scaffold formulation does not support cell migra-

tion; few cells should penetrate into the matrix or into

the central lumen of a cylindrical construct.

Cylindrical electrospun constructs were prepared andsutured shut on either end with 50 silk to form a hollow,

enclosed space (2 mm I.D. by 20–25 mm in length).

Adult Sprague Dawley rats (150–200 g) were anesthe-

tized to a surgical plane. Fur was removed from the

hindquarters and the skin was washed in betadine. Elec-

trospun constructs were implanted as hollow enclosed

cylinders. A representative example of an implant is

depicted in the inset of Fig. 1. The constructs wereimplanted into a blunt dissected channel prepared in

the belly of the vastus lateralis. Host muscle was sutured

380 T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385

over the implants and the skin incision was repaired.

Each of the electrospun materials described in this study

was implanted into three or four animals. Images pre-

sented in this preliminary study are representative of

the trials. Placement of a cylindrical construct into a

blunt dissected channel is illustrated in Fig. 1.For the recovery of implanted material, animals were

anesthetized to a surgical plane, the vastus lateralis was

dissected free of the surrounding tissue, trimmed of ex-

cess mass and immersion fixed in one-half strength Kar-

vonsky�s fix for 12 h. Excess tissue surrounding the

implanted materials was trimmed to define the outline

of the implants. Cylindrical constructs were identified

within the tissue, cut in cross-section, embedded and

Fig. 2. Synthetic polymer implants. Interface of electrospun PGA (A, B), P

seven days. Electrospun scaffolds of PGA failed to accumulate cells within t

open void in cross-section (CC in A, B). A fibrotic capsule (B, FC) was e

accumulation of multi-nucleated foreign body giant cells subjacent to the i

greater extent than the PGA-based implants and there was an accumulation o

arrowheads and inset panel D). Interstitial cells were scattered at low de

Electrospun cylinders composed of the PGA/PLA co-polymer blend exhibited

nearly devoid of cells (E, CC) and fibrotic capsules were evident at the hos

capsule; M, endogenous muscle of host. Size bar in Panel F represents 50 l

thick sections were prepared for light and ultrastructural

examination. For light microscopic images the tissue

was stained with toluidine blue/crystal violet solution.

3. Results

3.1. Implant testing

After seven days in vivo cylindrical constructs com-

posed of electrospun PGA delaminated from the sur-

rounding tissue of the host during recovery for

analysis. A fibrotic capsule was evident at the interface

of these implants and the surrounding tissue (Fig. 2A

LA (C, D) and PGA/PLA (E, F) based implants and host tissue after

he central lumen of the construct and this domain appeared as a large

vident at the interface of the implant and the host and there was an

mplant site (B, asterisks). PLA-based constructs were populated to a

f monlocular adipocytes within the central lumen of these constructs (C,

nsity throughout these tissue engineering scaffolds (D, arrowheads).

poor cellular infiltration, the central cores of cylindrical implants were

t implant interface (F, FC). CC, central core of implant; FC, fibrotic

m in panels A, C and E and 5 lm in panels B, D and F.

T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385 381

and B). Large rounded, multi-nucleated cells, consistent

in appearance with foreign body giant cells, were con-

centrated immediately subjacent to this fibrotic zone.

The central cores of these constructs appeared as hollow

spaces that were nearly devoid of cells. Constructs of

electrospun PLA lacked the pronounced fibrotic zonethat characterized the PGA-based implants and this

material appeared to support cell infiltration to a greater

extent. Interstitial cells and adipocytes were scattered at

low density throughout the electrospun PLA and within

the central domains of the implanted cylinders (Fig. 2C

and D). Scaffolds fabricated with electrospun PGA/PLA

co-polymer delaminated from the host tissue at recov-

ery. These constructs exhibited fibrotic capsules andaccumulated foreign body giant cells at the interface of

the implant site and the surrounding tissue of the host

(Fig. 2E and F). Few cells were evident within the hol-

low central cores of the PGA/PLA co-polymer based

scaffolds.

In contrast to scaffolds composed of the electrospun

biocompatible synthetic polymers, constructs composed

of electrospun type I collagen were fully infiltrated withinterstitial cells within seven days of implantation (Fig.

3A–C). At recovery, these implants were well integrated

and could not be separated by microdissection from the

Fig. 3. Collagen and gelatin-based implants. Electrospun constructs compose

of implantation. The transition zone (Panel A, TZ) occupied by the electrosp

continuum of cells. The central cores of the collagen-based implants were com

blood vessels were evident throughout these implants (B, arrows). Cells with

local axis of the electrospun collagen. Ultrastructural examination revealed ce

bundles of electrospun collagen fibrils that were present within the walls of t

were sparsely populated, developed fibrotic capsules and appeared to be infiltr

bars: In panel A = 100 lm, B = 50 lm and panel C = 5 lm, D = 50 lm.

surrounding muscle tissue of the host. There was a

smooth continuum of cells from the host tissue into

the electrospun collagen and no evidence of fibrotic

encapsulation. The central cores of implants composed

of electrospun collagen were completely filled with cells.

Functional blood vessels were scattered throughoutwalls of these implants.

To determine if the biological properties of electro-

spun collagen arise from the chemical composition of

the fibrils or other features that might be unique to this

native protein polymer we prepared constructs of elec-

trospun calfskin gelatin. Gelatin that is isolated from

calfskin is identical in chemical composition to acid sol-

uble collagen. However, during the processes used toisolate and prepare gelatin for commercial distribution

it is heat denatured and partially digested, events that

markedly alter the biological properties of collagen

[17,18]. After seven days in vivo cylindrical constructs

composed of electrospun gelatin delaminated from the

host tissue at recovery. Microscopic examination re-

vealed these implants were encapsulated and poorly

infiltrated with interstitial cells. The bulk of the cellspresent in these constructs had very little cytoplasm, a

histological feature characteristic of the lymphocyte

(Fig. 3D).

d of electrospun type I collagen were fully infiltrated within seven days

un matrix at the interface of the implant and host exhibited a smooth

pletely filled with a mixed population of cells (A, asterisks). Functional

in the wall of the cylindrical implants were aligned in parallel with the

lls (C, asterisks) within the scaffolds were fully intercalated between the

he implants (C, arrows). In contrast, constructs of electrospun gelatin

ated by lymphocytes (D, arrows). M = endogenous muscle of host. Size

Fig. 4. Representative scanning electron micrographs of electrospun materials. As judged by SEM tissue engineering scaffolds composed of

electrospun PGA (A), PLA (B), PGA/PLA co-polymer (not shown), collagen (C) and gelatin (D) have a similar appearance. We were unable to detect

any obvious structural properties that could be used to distinguish the electrospun materials with this type of analysis. Each matrix was composed of

discreet individual fibrils of similar cross-sectional diameter that were deposited as a non-woven scaffold. All images captured at 2000·, bar in each

image represents 10 lm.

382 T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385

As judged by scanning electron microscopy each of

the materials examined in this study has a similar

appearance when processed by electrospinning into a

fibrous tissue engineering scaffold (Fig. 4). However,

the PGA, PLA, PGA/PLA co-polymer, collagen andgelatin based materials induced very different host vs.

graft responses. The data from our implant studies sug-

gest that the biological properties of these materials are

not solely determined by the chemical identity of the

electrospun fibril. For example, by manipulating our

electrospinning conditions it was possible to produce

scaffolds of electrospun acid soluble collagen and elec-

trospun gelatin that exhibited a similar distribution offiber diameters (Fig. 5A) and pore properties (Fig.

5B). When tissue engineering scaffolds of electrospun

collagen were implanted, this fibrous matrix was rapidly

infiltrated by interstitial cells. Scaffolds composed of

electrospun gelatin induced fibrosis and accumulated

lymphocytes.

A cursory examination reveals at least three charac-

teristics that can be used to distinguish scaffolds of elec-trospun collagen from scaffolds of electrospun gelatin.

First, morphometric analysis indicates that subtle differ-

ences exist in the porosity of the different materials. The

average pore dimension of a matrix composed of elec-

trospun gelatin is consistently smaller than the average

pore dimension observed in a matrix of electrospun col-

lagen (Fig. 5B). However, we note that there is consider-

able overlap in the range of values that we determined

for this physical property. Second, electrophoretic anal-

ysis reveals, and confirms, that samples of electrospun

gelatin are composed of fragments of type I collagen(Fig. 5C). In comparison, samples of electrospun colla-

gen appear to be composed substantially of intact

monomers. Finally, at the ultrastructural level, fibrils

of electrospun collagen exhibit a 67 nm repeat banding

pattern [11] that is believed to expose a binding site in

the native collagen fibril that enhances cell adhesion

and migration [17,19,20]. In contrast, fibrils of electro-

spun gelatin lack this 67 nm repeat pattern and exhibita non-descript amorphous structure when examined by

transmission electron microscopy (Fig. 5D and E).

4. Discussion

Classically, the extracellular matrix has been viewed

as a static structural element that defines the three-dimensional architecture of an organ. However, there is

evidence of a dynamic interaction between the constitu-

ents of the extracellular matrix and the cellular

elements of an organ. Changes in the local microenviron-

ment can modulate the composition of the interstitium.

In turn, changes in the composition and organization

Fig. 5. Biophysical characterization of electrospun collagen and electrospun gelatin. Scaffolds of electrospun acid soluble collagen and gelatin exhibit

overlapping fiber (A) and pore dimensions (B) over a broad range of electrospinning conditions. Box plot illustrates the mean and range of values for

fiber diameter (A) and pore dimensions (B) observed in scaffolds of type I collagen and gelatin prepared by electrospinning. Values outside the

normal range are illustrated by dots. In this analysis average fiber diameter varied as a function of initial the electrospinning conditions (i.e. initial

concentration of polymer present); pore areas were more consistent over this same range of conditions. We note that average fiber diameter and pore

dimension are more uniform when collagen and gelatin are electrospun at low starting concentrations (40 mg/ml); these scaffold characteristics

become progressively less uniform as the starting electrospinning concentration increases (100 mg/ml). Electrophoretic analysis reveals that fibrils of

electrospun gelatin are composed of protein fragments (Panel C). Lanes 1–3 depict samples of electrospun collagen separated by SDS electrophoresis:

lane 1 = 2 lg protein/lane, lane 2 = 4 lg protein/lane, lane 3 = 10 lg protein/lane. Lanes 4–6 depict samples of electrospun gelatin. Lane 4 = 2 lgprotein/lane, lane 5 = 4 lg protein/lane, lane 6 = 10 lg protein/lane. Note that samples of electrospun collagen are composed of substantially intact,

large molecular weight monomers that appear as discreet protein bands. In comparison, lanes containing samples of electrospun gelatin show

smearing, evidence of protein fragmentation. Ultrastructural examination reveals that electrospun collagen (D) exhibits the 67 nm repeat pattern of

the native collagen fibril. Fibrils of electrospun gelatin exhibit an amorphous appearance at the ultrastructural level (F). Size Bars in panels C and

D = 100 nm.

T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385 383

of the extracellular matrix can regulate the phenotype,

and functional properties, of the cellular compartment

[16,21]. We believe this dynamic interaction effectively re-

stricts the range of materials that can be used in many

applications as tissue engineering scaffolds.

Under the conditions used in this study, electrospun

PGA, PLA, PGA/PLA co-polymer, gelatin and type Icollagen each deposit into tissue engineering scaffolds

composed of fibrils less than 1 lm in diameter. These

scaffolds exhibit similar average pore areas, ranging

from 2000 to 6000 lm2, with an average pore dimension

(distance between adjacent fibers) of less than 10 lm in

diameter for most scaffolds. These dimensions are well

below the size limit that normally suppresses the infiltra-

tion of cells into many types of biomaterials [22]. The

fibrotic capsules that developed in association with elec-

trospun PGA and the PGA/PLA co-polymer investi-

gated in this study are consistent with materials that

physically exclude cells from penetration. This structural

barrier may exist at the time the constructs are preparedand implanted or may evolve over time as these syn-

thetic materials biodegrade. Compounding any struc-

tural barriers that might inhibit the migration of cells

into a scaffold composed of these synthetic materials,

PGA and PLA can both release hydrolytic by-products

that can alter the local pH and cause an inflammatory

384 T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385

response [23]. The foreign body giant cells present in

association with these materials are clear evidence of im-

mune surveillance. Without further processing to cir-

cumvent these biophysical limitations, the structural

and chemical characteristics of scaffolds composed

solely of the synthetic polymers make them unsuitablefor many tissue engineering applications [24].

Collagen has long been recognized as a potential can-

didate material for tissue engineering applications.

However, conventional processing strategies have been

unable to recapitulate the structural and biological

properties of this natural polymer in a tissue engineering

scaffold. The process of electrospinning appears to rep-

resent a unique fabrication strategy to achieve theseobjectives. Scaffolds composed of electrospun, acid solu-

ble type I collagen are fully, and rapidly, infiltrated by

interstitial fibroblasts and microvascular endothelial

cells. Functional capillaries are present in this type of

scaffold within one week of implantation.

Scaffolds composed of electrospun gelatin that are

identical in chemical composition (i.e. calfskin type I

collagen isolates) and matched to the fiber diameter ofa matrix of electrospun collagen provoke inflammation,

initiate fibrosis and do not support cellular infiltration.

We have observed similar results in a guinea pig model

in which we have used electrospun acid soluble collagen

and electrospun gelatin to repair full-thickness dermal

injuries (not shown).

We noted three differences that could be used to dis-

tinguish a matrix composed of electrospun collagenfrom a matrix composed of electrospun gelatin. The dis-

tribution of fiber diameters and pore dimensions ob-

served in each scaffold differs to a subtle degree. We

have regulated fiber cross-sectional diameters to range

from 500 to 1000 nm for each of the materials examined

in this study. For collagen, the average pore dimension

for a matrix composed of fibers in this size range was

approximately 2000–6000 nm2. A matrix of gelatin withsimilar fiber diameters exhibited slightly smaller average

pore dimensions of 1500–4000 nm2. It is possible that

this somewhat smaller average pore dimension inhibits

the infiltration of cells into this material. However, as

noted, there is considerable overlap in the measured

pore diameters observed in these data sets. As a result

we do not believe the absolute porosity of these materi-

als represents a critical factor in determining the extentto which cells will infiltrate these scaffolds. Next, electro-

phoretic analysis revealed that fibrils of electrospun gel-

atin are composed of complex mixture of protein

fragments. This was evident in this analysis by a back-

ground of protein smearing and the lack of clear band-

ing in the SDS gel lanes. Conventional preparations of

gelatin (collagen gel) have a high inflammatory poten-

tial; peptide fragments released from denatured collagencan directly and indirectly induce the inflammatory cas-

cade [18]. Protein fragments released from tissue engi-

neering scaffolds composed of electrospun gelatin can

be expected to have similar pro-inflammatory activity.

Finally, electrospun type I collagen fibrils exhibit a 67

nm repeat structure. We argue that this topological fea-

ture plays a central role in allowing cells to freely pene-

trate an electrospun matrix of collagen.The VEGF-induced migration of microvascular

endothelial cells [20] and the infiltration of dermal fibro-

blasts [25] into collagen appear to be dependent upon an

a2b1 integrin binding site associated with the banded, 67

nm repeat pattern of collagen [17]. In keratinocytes, the

ligation of the a2b1 integrin induces the activation

of matrix metalloproteinase activity, an event that must

occur before these cells can effectively migrate on colla-gen [26]. Together, these observations suggest that scaf-

folds of electrospun collagen support rapid cellular

infiltration because this material can initiate, or potenti-

ate, physiological signals that promote cell migration.

The synthetic polymers of PGA, PLA and PGA/PLA

co-polymer lack specific integrin-binding sites and are

not directly subject to enzymatic attack by matrix metal-

loproteases or other proteolytic enzymes. In scaffolds ofelectrospun gelatin, the a2b1 integrin binding site (and

other binding sites) may be cryptic or, at best, present

at very low concentrations. The average pore dimension

of an inert, static tissue engineering scaffold, that lacks

physiologically relevant integrin binding sites, can be ex-

pected to represent a rate limiting step in the migration

of cells on this type of material.

From a materials processing standpoint, electrospin-ning is rapid and efficient. Nanoscale fibrils can be

deposited on a target mandrel in a dry, sterile state. This

type of tissue engineering material can be expected to

have an extended shelf life, an important consideration

in the commercial distribution process. The material

and chemical properties of an electrospun matrix can

be regulated at several different sites. For example, fiber

diameter and average pore dimension can be controlledby regulating the concentration of the materials present

in the starting electrospinning solutions [12,15]. Seam-

less and complex three-dimensional shapes can be pro-

duced [28] and by depositing fibrils of electrospun

collagen along a defined axis the alignment and distribu-

tion of cells within a bioengineered organ can be theo-

retically regulated to a high degree. This characteristic

has direct implications in the fabrication and functionof many different tissues; including skeletal muscle

[14], cardiac muscle [16] and smooth muscle-based or-

gans [27,28]. By supplementing electrospun collagen

with other matrix constituents, growth factors and/or

other peptides, the biological properties of a matrix

can be exquisitely tailored to a specific tissue or bioengi-

neering application [13,15]. These characteristics pro-

vide enormous flexibility to the tissue engineeringprocess. As a derivative of a natural, highly conserved,

and non-immunogenic extracellular matrix protein, elec-

T.A. Telemeco et al. / Acta Biomaterialia 1 (2005) 377–385 385

trospun collagen clearly has potential uses in the thera-

peutic delivery of stem cells [14], vascular bioengineering

[27,28], hard and soft tissue reconstruction, wound care,

and drug delivery [13].

Acknowledgements

This work supported in part by the Department of

Defense contract DAMD17-00-1-0512 (Simpson), NIH

R01EB003087 (Simpson), NanoMatrix Inc. (Simpson,

Bowlin and Wnek) and the Whitaker Foundation (Bow-

lin). The authors thank John Povlishock, Ph.D. for com-

ments, Cynthia Allen and Robert Wise for surgical andtechnical expertise, Judy Williams for ultrastructural

analysis and the Technology Transfer Office of Virginia

Commonwealth University for support. US and Inter-

national Patents Issued and Pending.

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