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Biointeractive polymers and tissue engineered blood vessels
Biomoteriols 17 (1996)
329-336
Elsevier Science Limited Printed in Great Britain. All rights reserved 0
1996
0142.9612/96/$15.00
Biointeractive polymers and tissue engineered blood vessels * Howard P. Greislertt , Claire Gosselint , Dewei Rent, Steven S. Kangt and Dae Un Kim § tLoyo/a University Medical Center, Department of Surgery, 2760 South First Avenue, Maywood, IL 60153, USA; 1:Hines VA Hospital, Hines, IL 60141, USA; 5 St. Barnabas Medical Center, Livingston, NJ 07039, USA The regulation
of endothelial
interventions
is critical
biomaterials
with suspensions
modulating reported
cell (EC) and smooth
to clinical
efficacy.
containing
EC and SMC growth that 60~ internodal
and heparin
develop
after 4 weeks
in dogs.
thicker
(139p)
inner
effects
of FGF-1, heparin,
bioactive
in vitro
distance
confluent
(P < 0.001) in DNA synthesis.Heparin with complete
1 within
FG without
increase
while
increasing
FGF-1 and heparin ‘> 3.2 U ml-’ between activity other
heparin
in the medium
stimulated
to higher
SMCs
to induce
by the applied
significantly
in vitro.
(versus
we studied
FG caused
P< 0.001). FGF-
and then decreased
effects.
was blocked
ratio.
This same
concentrations inhibition
the relative
system
or systemic
proliferation.
A synergism
EC growth
to modulate
protein
growth
Only thrombin
by heparin.
but without
the
a 182%
in a dose-dependant
FG alone,
increased
It is thus possible
the FGF:heparin
local affects
developed
a dose-dependant
had similar
FGF-1
EC and SMC proliferation
FGF-1 caused
was also found
of heparin.
by altering
other
SMCs
and this stimulation
on EC proliferation
concentrations
of ECs versus proteins
of quiescent
initially
glue (FG) containing
this effect
at 500U ml-’
but together,
concentrations
SMC growth
FGF and heparin
response
had no effect,
FG diminished
growth
previously
SMC proliferation
on SMC growth
within
of FG-induced
of differentially
We have
increased were
vascular
of impregnating
in the capability
with fibrin
after 20 weeks
concentrations
manner,
heparin
impregnated
following
a method
implantation.
to the FGF. To minimize
increase
proliferation
resulting
with transiently
implants
in response
inhibition
proteins
ePTFE grafts
endothelialization
and thrombin
cell (SMC)
has developed
and in viva following
Thoraco-abdominal
capsules
muscle
Our laboratory
in
proliferative
may be useful
affects
following
with
release
of that protein. Keywords:
Tissue
engineering,
vascular
grafts,
FGF, EC, SMC, heparin
Received 12 December 1994; accepted 17 February 1995
many investigators to pursue strategies aimed at optimizing the biological interaction with the synthetic polymer rather than to minimize all biological interaction. Thus, the term ‘biocompatible’ has had a subtle change in its definition from a relative lack of biological response to a relatively desirable set of biological responses. The implanted polymer instantaneously affects the surrounding biological milieu, resulting in alterations in protein structure, platelet activation, inflammatory response, etc. By the same token, these biological responses necessarily alter the surface and to some extent bulk properties of the polymer and thus a continuous dynamic is achieved by which both the biological milieu and the polymer each alter the behaviour of the other. This results in the establishment of a novel ‘prosthesis-tissue complex’ whose properties differ from either component alone. Recent research has led to some understanding of how specific chemical and/or biomechanical parameters of the polymer may alter a variety of biological responses including the generation of growth factors by locally recruited
Research aimed at the development of more clinically efficacious small-diameter vascular grafts has been profoundly influenced by two major conceptual advances in our understanding of arterial wall biology. When Voorhees et a1.l reported the first successful application of a synthetic vascular graft, in this case made of Vinyon N, the arterial wall was considered to be a simple conduit through which blood passed. Since that time, however, an extraordinary expansion of our knowledge of arterial wall biology has documented an exceedingly complex set of metabolic activities by arterial wall cells. This advance has led to a more sophisticated approach to research in arterial replacement materials. A concomitant advance has occurred in the field of biomaterials. The long sought after biologically inert material has not as yet been developed, and the realization of the inherent ‘biointeractivity’ of all implanted polymers has led *Presented at the Biomedical Engineering October 1994. Correspondence to Dr H.P. Greisler.
Society
Meeting,
329
Biomaterials
1996. Vol. 17 No. 3
330
Biointeractive
polymers
cells, resulting in dramatic alterations in the degree of smooth muscle cell, myofibroblast and endothelial cell recruitment to the implanted bloodcontacting biomaterial. We and others have pursued a series of investigations demonstrating the dramatic difference in mesenchymal tissue incorporation induced by different polymeric materials and documenting the role of locally secreted growth factors in response to these implants as mediators of these different healing responses. For example, the implantation of lactide-glycolide copolymerit biomaterials induces local macrophages to synthesize, among other proteins, basic fibroblast growth factor (FGF-2), resulting in a significantly thicker and more cellular inner capsule as compared to either Dacron or expanded polytetrafluoroethylene (ePTFE) materials. Using this observation, we have pursued studies of methods of applying exogenous fibroblast growth factor to ePTFE to induce similar mesenchymal tissue ingrowth. When vascular prostheses are constructed of either polyglycolic acid (PGA) or of Dacron to nearly identical weave characteristics, wall thickness, including porosity and elastic modulus, the tissue ingrowth following implantation of these two materials differs greatly. Serial explants at time intervals ranging from 2 weeks to 1 year following implantation into rabbit i&arena1 aortas (48 PGA and 21 Dacron) were studied by gross and histological techniques”,“. The PGA material was resorbed between 4 weeks and 3 months following implantation. No rabbit displayed evidence of haemorrhage, perigraft haematoma, false aneurysm or graft infection. The PGA explants displayed a 10% incidence of aneurysmal dilatation. Inner capsule thickening increased significantly in the PGA group from 2 weeks until 2 months, at which time it stabilized at 3.17 times greater than that reached in the Dacron control group. Differences in tissue thickness resulted from differences in cell numbers and extracellular matrix content. Histologically, by 2 weeks macrophages and foreign body giant cells infiltrated the PGA interstices and inner capsules, and intracellular PGA inclusions were found as evidence for phagocytosis. By contrast, these inflammatory cells abutted the Dacron material but without evidence of intracytoplasmic Within 2 weeks of the appearance of inclusions. phagocytosis of PGA by macrophages, the inner capsules dramatically thickened with numerous circumferentially and longitudinally oriented layers of myofibroblasts amidst densely packed collagen, as shown in the 4 week PG910 explant represented in Figure lb and controlled with the 4 week Dacron explant shown in Figure la. These cells stained positively with an anti-sc-actin antibody and were covered along the blood-contacting surface by a confluent layer of Factor VIII positive endothelial cells, which by transmission electron microscopy were demonstrated to contain Weibel-Palade bodies. By contrast, at this same 4 week time point the inner capsule within Dacron prostheses remained a fibrin coagulum with minimal mesenchymal cell incorporations. By 3 months the PGA was fully resorbed, the macrophage population gone, and the neointima and flow surface changed little from 3 to 12 months.
and tissue
engineered
blood
vessels:
HP.
Greisler
et al.
inflammatory
Biomaterials 1996. Vol. 17 No. 3
Figure 1 Top: mid-portion of a Dacron specimen explanted after 1 month showing inner capsule (IC) composed of fibrin coagulum and minimal cellular repopulation (haematoxylineosin, original magnification x74). Bottom: mid-portion of 1 month polyglycolic acid specimen showing greatly thickened IC of circumferentially and longitudinally orientated smooth muscle-like myofibroblasts beneath endothelial-like flow surface. Progressively more peripheral to the lumen are the prosthesis and the well-vascularized outer capsule (haematoxylin-eosin, original magnification x74). (Reproduced from Ref. 3, by permission of the American Medical Association.)
Slower resorption produced by an increase in the ratio of 1actide:glycolide rings resulted in a similar but more slowly induced tissue ingrowth. This altered chemistry was used in production of polydioxanone (PDS) grafts. Twenty-eight PDS prostheses were implanted into rabbit aortas and harvested from 2 weeks to 1 year4. Among these explants, mild aneurysmal dilatation was found in l/28 and no stenoses or occlusions were encountered. The rate of tissue ingrowth paralleled the kinetics of macrophage mediated prosthetic resorption, with progressive inner capsule thickening ending after 3-6 months at 420 pm, a thickness not statistically different from the maximum of 480 pm found after 2 months in the PGA specimens. Capillarization of the inner capsules apparently resulting from a transinterstitial ingrowth from the surrounding tissue was found to communicate with the blood-contacting surface and was considered the source of the confluent endothelial layer along that surface (Figure 2). Following explantamechanical tion, studies demonstrated these regenerated tissue conduits to display arterial-like elasticity characteristics and resistance to fatigue or bursting at 6000 mmHg (-798MPa) peak systolic and 2000mmHg (-266 MPa) mean pressures. Freshly
Biointeractive
Figure 2
polymers
Mid-portion
and tissue engineered
blood vessels: HP. Greisler etal.
than those found within prostheses similarly woven of yarns containing 100% PG910. Support for the observation that inner capsule tissue ingrowth within bioresorbable prostheses results from a transinterstitial migration came from analyses of compound prostheses in which a central PG910 segment (10 mm long) was placed between proximal and distal Dacron segments (10 mm) and implanted into rabbit i&arena1 aortas. These grafts were explanted in triplicate. The inner capsule thickness within the PG910 segments increased from 2 weeks to 2 months and was significantly thicker than either Dacron segment at both 1 and 2 months (P < 0.004 and P < 0.0001,respectively). At these time points the inner capsules within the Dacron segments remained devoid of mesenchymal tissue beyond 2 mm of pannus ingrowth. The junctions between the inner capsules of the two materials were abrupt. Inner capsule thickening results not only from cell migration and extracellular matrix deposition but prominently from proliferation of ingrowing endothelial cells and myofibroblasts. Mitotic activity within inner capsules was studied using autoradiographic techniques to demonstrate incorporation of tritiated thymidine into newly synthesized DNA. PG910, PDS and Dacron prostheses were implanted into the infrarenal aortas of rabbits and specimens harvested from 2 weeks to 3 months following implantation. Autoradiographic analyses on crossadministration of following tritiated sections thymidine demonstrated a significant increase in mitotic index within the two bioresorbable groups, the rate of cell proliferation paralleling the course of prosthetic resorption (Table l)fi. PG910 explants showed a mitotic index of 28.3 + 23.2% 3 weeks after implantation, this proliferative activity progressively decreasing to 1.2 + 1.1% after 12 weeks. The more slowly resorbed PDS group demonstrated a more prolonged elevation of mitotic activity, 7.5 & 5.6% after 12 weeks. The Dacron group, by contrast, never exceeded proliferation rates greater than 1.2 & 0.9%. The location of the labelled proliferating cells was deep in the inner capsules in proximity to the macrophages and prosthetic material. In this zone the myofibroblasts demonstrated the synthetic ultrastructural phenotype commonly displayed by actively cycling cells”. Similarly, collagen content within the inner capsules parallels the kinetics of prosthetic resorption7. Collagen content measured by hydroxyproline calorimetry within the inner capsules 1 month following implantation of PG910 prostheses measured
of PDS specimen at 2 months showing
capillary invading inner capsule and communicating with luminal surface. Endothelialized luminal surface of specimen appears to be continuous with capillary wall (haematoxylineosin, original magnification x268). (Reproduced from Ref. 4, by permission of American Medical Association.)
explanted tissues were perfused and perfusates assayed for 6-keto-PGF,,/TxB, ratios before and after the administration of arachidonic acid. Results from tissues explanted at 6 and 12 months (1.62 i 0.70) did not statistically differ from control rabbit aortas (1.85
+I 0.61).
The construction of woven prostheses made from compound yarns containing both PGA or polyglactin 910 (PG910) plus Dacron components results in a significant diminution in tissue ingrowth and inner capsule cellularity”. The inner capsules within prostheses constructed of yarns containing 80% PG910 and 20% Dacron were statistically significantly thinner Table 1
Mitotic
331
index (%) PG910
PDS
Dacrona
2 3
1.42 f 0.59 28.34 f 23.21
No IC
No IC -
4 12 52
7.58 f 2.02* 1.17 f 1.05” 0.72 f 0.98””
7.50 * 2.66 7.50 i 5.59 1 .oo i 0.22****
No IC
Implant
aDue
to the
the other ‘P
duration
(weeks)
relativehypocellularlty
groups
< 0 01 versus
are
lmposslble
12 week
Dacron;
of the inner However, “P
c
the
capsules
(ICs)
hypocellularity
0 01 versus
4 week
prior itself PG910.
to 12 weeks
in the Dacron
is due largely *“P
group,
It 0.3v** 1.22 l 0.90*** 1.18
statistlcal
to the lack of mltot!c
<: 0 05 versus
4 week
PDS,
““P
activity
comparisons
in mltotlc
index
between
Dacron
and
in thts group
c 0 05 versus
4 week
PDS.
Biomaterials
1996.
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No. 3
Biointeractive
332
35.5 5 9.9 compared
polymers
pg (mg collagen))’ rng-’ dry weight to 25.6 i 5.0 in the PDS group and 12.0 * 0.9 in the Dacron group (P < 0.02 or P < 0.01 comparing PC910 or PDS to Dacron, respectively). The collagen content within the normal rabbit aorta measured 22.5 f 1.2 pg mgg. Based upon the temporal and spatial correlation between proliferating cells and macrophages, we that macrophage-biomaterial interhypothesized actions may yield a differential activation of the macrophage, inducing a differential secretion of bioactive factors either stimulatory or inhibitory to myofibroblast, smooth muscle cell and endothelial cell proliferation. We further hypothesized that this secretion of growth modulators played a major role in the initiation of the extensive mesenchymal cell ingrowth seen following implantation of lactideglycolide copolymeric bioresorbable prostheses. To test these hypotheses, unstimulated peritoneal macrophages harvested from New Zealand White rabbits and identified by immunoperoxidase staining using the rabbit macrophage specific RAM 11 antibody were cultured in vitro. To the culture media were added particles of either PG910, Dacron or neither material. Conditioned media were assayed for mitogenic activity using growth assays of quiescent BALB/c 3T3 cells, rabbit aortic smooth muscle cells and LE-II murine capillary lung endothelial cells. Macrophages exposed in vitro to PG910 released into their culture media substances which induced significantly more DNA synthesis in BALB/c 3T3 cells (1.6-3.3 times) as compared to macrophages exposed to Dacron or to neither biomaterial. Similar results were found in bioassays using smooth muscle cells and endothelial cells (Figure 3)‘. The greatest mitogenic activity within the conditioned media of macrophages exposed to PG910 was encountered during weeks 3-5 of culture, a time corresponding to visually apparent PG910 phagocytosis by the cultured cells.
MACROPHAGE
CONDITIONED
RABBIT
SMC
MEDIA
STUDY
BIOASSAY
Figure 3 Tritiated thymidine incorporation into normal rabbit aortic smooth muscle cells exposed to conditioned media collected weekly from normal rabbit macrophages cultured in the presence of PG910, Dacron or neither biomaterial (mean fs.d., P < 0.05 for PG910 versus control weeks 1-5, and Dacron versus control weeks 1 and 2). Biomaterials
1996, Vol. 17 No. 3
and tissue
engineered
blood
WCKOPHAGE
vessels:
CONDIIIONED RABBIT
NE,‘TRAI
SMC l7lNG
HP.
MEL’IA
Greisler
ef al.
STUDY
BIOASSAY AhTImbiir
Bt,
Figure 4 New Zealand White rabbit aortic smooth muscle cell bioassay using conditioned media from macrophages harvested from rabbits and exposed for l-7 weeks to either no biomaterial, polyglactin 910 or Dacron. The solid bars represent the results in the absence of the neutralizing anti-basic FGF antibody and the patched bars represent the results following the pre-incubation of conditioned media with a concentration of neutralizing anti-basic FGF antibody equal to five times the NDSO (mean f sd.).
The smooth muscle cell bioassays were repeated following incubation of the conditioned media with a neutralizing anti-basic FGF antibody (Upstate Biotechnology, Lake Placid, NY, USA). The two- to three-fold increase in smooth muscle cell proliferation induced by macrophage exposure to PG910 was diminished by 50-80s (Figure 4). A similar reduction in mitogenicity of conditioned media of macrophages exposed to Dacron was induced by this neutralizing antibody. By contrast, no change in mitogenicity of media of macrophages in the absence of biomaterial was found. These results corresponded to Western blot analyses showing immunoreactivity with an antibody against FGF-2 in the conditioned media of macrophages exposed to either biomaterial but no immunoreactivity of the media of macrophages in the absence of biomaterials. Parallel experiments evaluating the presence of transforming growth factor-a (TGF-/I) in conditioned media suggested that macrophages exposed to Dacron released into the media significantly greater amounts of bioactive TGF-P as compared to macrophages not exposed to biomaterialsg,lO. This may be interpreted either as a greater synthesis and/or secretion of active TGF-P, [as shown by neutralization of activity by preincubation with a neutralizing anti-TGF-/?, antibody), or conversely, as a greater degree of activation of latent TGF-P, the degree of secretion of which may or may not have been altered by interactions between the macrophages and Dacron. The studies described above suggest that different biomaterials may induce dramatically different degrees of smooth muscle cell, myofibroblast and endothelial cell ingrowth via a transinterstitial migration with a transient proliferative response, and that this is initiated at least in part by production of growth factors by activated macrophages. We next reasoned
Biointeractive
polymers
and tissue engineered
blood vessels:
that the application of specific growth factors to biomaterials may induce a similar response. In early studies published in 198611,‘2, we affixed FGF-1 (acidic FGF) to Dacron and to polydioxanone surfaces by sequential application of fibronectin followed by heparin (via its fibronectin affinity), FGF-1 (via its heparin affinity) and finally a second heparin layer because of the ability of heparin to protect FGF from proteolytic degradation as well as its synergistic activity with the growth factor. The retention of lz51FGF-1 by the preparation prosthesis following implantation into the rabbit aorta was quantitated. Following 1 week in circulation, grafts were explanted and FGF-1 eluted using salt solutions, and the FGF-1 was shown to have retained its molecular integrity as determined by polyacrylamide gel electrophoresis. This eluted FGF was shown to have retained its mitogenic activity in culture, inducing the anticipated DNA synthesis when added to quiescent murine capillary lung endothelial cells. However, using this FGF application technique, no enhancement of in vivo spontaneous endothelial cell ingrowth or proliferative activity of seeded endothelial cells was documented. Further in vitro studies led us to conclude that FGF-1 bound to immobilized heparin did not possess mitogenic activity until the bond either between the FGF-1 and heparin or between the heparin and fibronectin was broken. The ability of a variety of FGF delivery systems to promote endothelial cell growth was studied in a cell culture model. Surfaces pretreated with fibronectinl heparin/FGF-llheparin did not induce growth of human umbilical vein endothelial cells or canine jugular vein endothelial cells when soluble FGF-1 was excluded from the media. Using the same assay system, we screened FGF affixation techniques and found the binding of FGF-1 to any immobilized substrate rendered FGF-1 devoid of endothelial cell mitogenic activity. However, utilizing the same assay we developed a method currently employed in our laboratory in which FGF-1 and heparin are placed into suspension within a fibrinogen/thrombin matrix (fibrin glue)13. In vivo release kinetics were quantitated using fibrin glue suspensions containing 1251-FGF-1 and heparin impregnated into ePTFE grafts implanted into rabbit infrarenal aortas. After 7 and 30 days in circulation, 13.4 +6.9% and 3.8 f l.l%, respectively, of the applied 1251-FGF-1 remained on the prostheses. When calculated as percent changelmin change, the rate of loss during the initial 1 h averaged -24.1 A%lA min. After this initial hour the rate of loss from 60 min to 30 days averaged -0.03 A%lA min. The distribution of the 1251-FGF-1 across the wall of the prostheses was linear, with equal radioactivity found in each successive 1/18th segment of the 600 pm thickness of graft wall, documenting the application technique capable of distributing the FGF-1 along the inner as well as outer graft surfaces and throughout the internodal spaces. Utilizing this impregnation technique, we evaluated the effect of this fibrin glue/heparin/FGF-1 preparation on graft healing, endothelialization and endothelial cell proliferation in a bilateral canine aorto-iliac
HP.
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et al.
333
bypass graft model13. ePTFE grafts, 60 pm internodal distance, treated with fibrin glue/heparin/FGF-1 (Group I) were implanted contralateral to either ePTFE grafts treated with fibrin glue/heparin but devoid of FGF-1 (Group II) or untreated ePTFE grafts (Group III). Grafts were explanted after 7 or 28 days. At 28 days every Group I explant displayed a totally confluent Factor VIII positive endothelialized surface and extensive capillarization through the internodal spaces of the graft wall from the surrounding tissue. By contrast, no specimen of either Group II or Group III displayed a confluent endothelialized surface, and only occasional small islands of cellularity were observed interspersed with areas of fibrin coagulum populating the inner capsules. En face autoradiographic analysis of tritiated thymidine incorporation into newly synthesized DNA (Table 2) documented a significant increase in endothelial cell mitotic activity in the 28 day explants pretreated with fibrin glue containing heparin and FGF-1 (P < 0.05). Further studies were performed to evaluate the potential of this FGF-1 application technique to induce endothelialization over longer length grafts as well as the potential for this system to induce myointimal hyperplasia at later time points14. Thoraco-abdominal aortic implants (30 cm length x 8 mm i.d.) of 60 pm internodal distance ePTFE were again divided into the three groups described above. Grafts were explanted after 10, 30 or 140 days. All 26 explants were patent and none showed evidence of thrombus formation, stenosis or dilatation. No gross or histological evidence of anastomotic intimal hyperplasia was seen. Histologically, the untreated grafts were lined solely with fibrin coagulum through 30 days, whereas at 20 weeks small islands of pseudo-intima containing several layers of smooth muscle cells covered portions of the explants interspersed with areas of thrombus. The specimens pretreated with FGF-1 and explanted after 10 days showed an extensive mesenchymal tissue ingrowth into the body of the graft, with smooth muscle cells and capillaries extending 80% of the distance from outer to inner graft surface. By 30 days the entire graft was extensively infiltrated with abundant capillarization, and the blood-contacting surface was nearly 100% confluent and positive for the presence of Factor VIII by immunoperoxidase staining. This endothelialized neointima remained throughout the entire 30 cm graft length at 140 days, again with extensive capillarization throughout the graft wall (Figure 5A and B). The source of the capillary ingrowth was apparently a migration from the surrounding tissue. Along the outer surface, capillary
Table 2
Number of labelled cells per high-power field (x40)
Group I (FGa/heparin/FGF-1) Group II (FG/heparin) Group Ill (untreated) * FG : fibrin
7 days
28 days
3.01 * 1.95 1.72 ?c 1 .Ol 1.45 It 0.49
22.18 zt 12.87’ 2.42 & 1.65’ 1.09 f o.55c
glue.
bP
< 0.02
versus
Group
‘P
< 0.05
verws
Group
I (7 I (28
days). days)
Biomaterials 1996, Vol. 17 No. 3
334
Biointeractive
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A
and tissue
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Greisler et al.
B
Figure 5 A, Photomicrograph of an FG/FGF-l-treated graft explanted after 20 weeks showing a more developed inner capsule consisting of numerous layers of longitudinally orientated myofibroblasts and collagen and covered by an endothelial cell monolayer (haematoxylin-eosin. original magnification x724). 6, Photomicrograph of the same FG/FGF-l-treated graft showing abundant capillarization of the graft wall and two mitotic figures (arrows) (haematoxylin-eosin, original magnification x724)
infiltration of the internodal spaces (Figure 6A) could be seen at all time points in grafts pretreated with the FGF-1. As early as 10 days following implantation, capillaries could he seen extending through the graft wall and reaching the blood-contacting surface of the graft (Fi,our’r h‘B and C), with continuity of the endothelial cells between ingrowing capillaries and the blood-coIitac:ting surface of the implanted graft. This abundant mesenchymal tissue and capillary infiltration was accompanied by a transient FGF-1 induced increase in cell proliferation. Cross-sectional autoradiography (Fi,nure 7) showed at 30 days the effect of FGF-1 in inducing a proliferative response, with 9.6 i 4.4 of the cells picking up the tritiated following during the 10 h period thymidine thymidine administration. At 20 weeks the mitotic index had decreased in all groups, measuring less than 1% with no difference among the groups. The inner capsule thickness among 20 week explants, however, revealed a greater degree of thickening in the grafts pretreated with the FGF-1 (139 i 178 pm). No significant difference was seen comparing the peri-anastomotic and more central zones.
IJsing reverse transcription polymerase chain reaction techniques, we evaluated the expression by inner capsular cells of mRNA for FGF-1, FGF-2, TGFType I (flg) to ascertain B, and the FGF receptor whether the presence of exogenous FGF-1 might induce a secondary upregulation of other growth factor genes which might over time result in intimal hvperplasia’4. No difference was seen in the level of expression of these growth factors when comparing results between the three groups of grafts. Northern blot analysis for platelet-derived growth factor (PDGF) A and PDGF B chain message similarly showed no apparent difference in the level of expression as a function of either time or graft treatment method. Further in vifro studies in our laboratory have been designed to modify the FGF-1 : heparin ratio so as to solectivel! induce endothelial cell proliferation without a concomitant increase in smooth muscle cell exists proliferation”. A complex relationship between FGF-1 and heparin as concerns smooth muscle cell proliferation. Heparin synergizes with FGF-1 in stimulating the proliferative response of smooth muscle cells. whereas heparin in the absence
Biointeractive
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335
C Figure 6 A, Photomicrograph of an FG/FGF-l-treated graft implanted after 20 weeks showing the abluminal portion of the graft wall and adjacent outer capsule. Numerous capillaries, C, can be seen with some transversing the tissue-material interface and entering the graft’s internodal spaces (haematoxylin-eosin, original magnification x825). B, Photomicrograph of the same FG/FGF-l-treated graft as that shown in figure5A showing abundant capillarization, C, filled with red blood cells in the graft wall and showing a mitotic figure (arrow) (haematoxylin-eosin, original magnification x825). C, Photomicrograph of an FGIFGF-l-treated graft implanted after 30 days showing the luminal portion of the graft wall with a capillary, C, extending to the luminal surface and with endothelial cells (arrow) lining both the capillary and the luminal surface (haematoxylin-eosin, original magnification x825)
of FGF-1 inhibits smooth muscle cell proliferation. A synergism between FGF-1 and heparin in stimulation of endothelial cell proliferation occurs, but no heparin-induced inhibition of endothelial cell proliferation has been reproducibly documented. We thus hypothesized that there might exist a ratio of FGF-1 :heparin that selectively supports the growth of endothelial cells as compared to smooth muscle cells. In a series of in vitroanalyses, we evaluated the growth of both rabbit and canine arterial smooth muscle cells in response to either soluble FGF-1 and heparin or to FGF-1 and heparin within suspensions of fibrin glue, upon which the smooth muscle cells were cultured. Smooth muscle cell proliferation was significantly increased when either 5 or 50 Uml-’ of heparin (+85% and +66%, respectively, P < 0.0001 for both) was added to 10 ng ml-l of FGF-1. However, smooth muscle cell proliferation was significantly decreased from control (-IL?%, P= 0.014) by the addition of 500 U ml-l of heparin. Concomitant
studies on human umbilical vein endothelial cell proliferation showed a significant increase by the addition of 5, 50 or 500 U ml-’ heparin (+68%, +99% and +lOS%, respectively, P < 0.0001 for all). No significant inhibition of endothelial cell proliferation was found at even the highest, 500 UmlK’, heparin concentration studied. The effect of this FGF1: heparin ratio on selectively inducing endothelial cell proliferation without smooth muscle cell proliferation in vivois currently being evaluated. From these studies we conclude that different polymers, by their different biochemical characteristics and/or biomechanical properties, are capable of inducing dramatically different degrees of mesenchymal tissue ingrowth. The mechanism in part appears to be a differential for this difference activation by the polymers of macrophages with which they interact, resulting in a differential production of growth factors which may either stimulate or inhibit endothelial cell, smooth muscle cell and myofibroblast Biomaterials
1996,
Vol. 17 No. 3
Biointeractive
336 CROSS
SECTIONAL TRITIATED
polymers
and tissue
THYMIDINE
2 3
4
5
6
7 140
DA”;
Figure 7 Mitotic index expressed as number of labelled cells per 100 cells within the inner capsules (mean + s.e.m.).
8
9
proliferation and migration. A similar alteration in ingrowth of these cells may be induced by application of exogenous growth factors when impregnated into prosthetic materials in a biologically active delivery system. The ideal polymers and the ideal growth factors as well as the ideal delivery systems have certainly not yet been determined. We contend, however, that by use of these techniques the biological response to implanted biomaterials may be selectively regulated so as to result in the regeneration of functioning arterial conduits following implantation of partially bioresorbable or either bioresorbable, relatively inert but non-resorbable biomaterials.
10
11
ACKNOWLEDGEMENTS Supported in part by grants from the National Institutes of Health, numbers ROl HL29268 and ROl HL41272. The authors wish to acknowledge Mr Richard Dewitt for the preparation of the photomicrographs, MS Jacqueline Garfield for the electron microscopy preparation and Peter I. Kim for his assistance in the electron microscopic analyses.
3
vessels:
HP.
Greisler
et al.
Voorhees AB, Jaretzki A, Blakemore AH. The use of tubes constructed from Vinyon N cloth in bridging arterial defects. Ann Surg 1952; 135: 332. Greisler HP. Arterial regeneration over absorbable prostheses. Arch Surg 1982; 177: 1425-1431. Greisler HP, Kim DU, Price JB, Voorhees AB. Arterial regenerative activity after prosthetic implantation. Arch Surg 1985; 120: 315-323. Greisler HP, Ellingher J, Schwartz TH, Golan J, Raymond RM, Kim DU. Arterial regeneration over polydioxanone prostheses in the rabbit. Arch Surg 1987; 122: 715-721. Greisler HP, Schwartz TH, Ellinger J, Kim DU. Dacron inhibition of arterial regenerative activity. 1 Vast Surg 1986; 3: 747-756. Greisler HP, Petsikas D, Lam TM, Pate1 N, Ellinger J, Cabusao E, Tattersall CW, Kim DU. Kinetics of cell proliferation as a function of vascular graft material. J Biomed Mater Res 1993; 27: 955-961. Cabusao EB, Lam TM, Ellinger J, Greisler HP. Kinetics of collagen deposition within bioresorbable and nonresorbable vascular prostheses. Tram Am Sot Artif Organs (ASAIO) 1991; 20: 113. Greisler HP, Ellinger J, Henderson SC, Shaheen AM, Burgess WH, Lam TM. The effects of an atherogenic diet on macrophage/biomaterial ineractions. J Vast Surg 1991; 14: 10-23. Petsikas D, Cziperle DJ, Lam TM, Murchan P, Henderson SC, Greisler HP. Dacron-induced TGF-8 release from macrophages: effects on graft healing. Surg Forum 1991; 42: 326-328. Greisler HP, Petsikas D, Cziperle DJ, Murchan PM, Henderson SC, Lam TM. Dacron stimulation of macrophage TGF-P release. Cardiovasc Surg 1994; (in press). Greisler HP, Klosak JJ, Dennis JW, Ellinger J, Kim DU, Burgess WH, Maciag T. Endothelial cell growth factor (ECGF) attachment to biomaterials. ASAIO Tram 1986;
32: 346-349. 12
13
1996, Vol. 17 No.
blood
REFERENCES
AUTORADIOGRAPHY
1
Biomaterials
engineered
14
15
Greisler HP, Klosak JJ, Dennis JW, Karesh SM, Ellinger J, Kim DU. Biomaterial pretreatment with ECGF to augment endothelial cell proliferation. J Vast Surg 1987; 2: 393402. Greisler HP, Cziperle DJ, Petsikas D, Murchan PM, Appelgren EO, Drohan W, Burgess WH, Kim DU. Enhanced endothelialization of expanded PTFE grafts by heparin binding growth factor-type I (HBGF-1). Surgery 1992; 112: 244-255. Gray JL, Kang SS, Zenni GC et al. FGF-1 affixation stimulates ePTFE endothelialization without intimal hyperplasia. J Surg Res 1994; 57: 596-612. Kang SS, Ren D, Gosselin C, Greisler HP. Smooth muscle cell inhibition with endothelial cell stimulation by growth regulators within a fibrin glue delivery vehicle. Surg Forum 1994; 45: 355-358.
329-336
Elsevier Science Limited Printed in Great Britain. All rights reserved 0
1996
0142.9612/96/$15.00
Biointeractive polymers and tissue engineered blood vessels * Howard P. Greislertt , Claire Gosselint , Dewei Rent, Steven S. Kangt and Dae Un Kim § tLoyo/a University Medical Center, Department of Surgery, 2760 South First Avenue, Maywood, IL 60153, USA; 1:Hines VA Hospital, Hines, IL 60141, USA; 5 St. Barnabas Medical Center, Livingston, NJ 07039, USA The regulation
of endothelial
interventions
is critical
biomaterials
with suspensions
modulating reported
cell (EC) and smooth
to clinical
efficacy.
containing
EC and SMC growth that 60~ internodal
and heparin
develop
after 4 weeks
in dogs.
thicker
(139p)
inner
effects
of FGF-1, heparin,
bioactive
in vitro
distance
confluent
(P < 0.001) in DNA synthesis.Heparin with complete
1 within
FG without
increase
while
increasing
FGF-1 and heparin ‘> 3.2 U ml-’ between activity other
heparin
in the medium
stimulated
to higher
SMCs
to induce
by the applied
significantly
in vitro.
(versus
we studied
FG caused
P< 0.001). FGF-
and then decreased
effects.
was blocked
ratio.
This same
concentrations inhibition
the relative
system
or systemic
proliferation.
A synergism
EC growth
to modulate
protein
growth
Only thrombin
by heparin.
but without
the
a 182%
in a dose-dependant
FG alone,
increased
It is thus possible
the FGF:heparin
local affects
developed
a dose-dependant
had similar
FGF-1
EC and SMC proliferation
FGF-1 caused
was also found
of heparin.
by altering
other
SMCs
and this stimulation
on EC proliferation
concentrations
of ECs versus proteins
of quiescent
initially
glue (FG) containing
this effect
at 500U ml-’
but together,
concentrations
SMC growth
FGF and heparin
response
had no effect,
FG diminished
growth
previously
SMC proliferation
on SMC growth
within
of FG-induced
of differentially
We have
increased were
vascular
of impregnating
in the capability
with fibrin
after 20 weeks
concentrations
manner,
heparin
impregnated
following
a method
implantation.
to the FGF. To minimize
increase
proliferation
resulting
with transiently
implants
in response
inhibition
proteins
ePTFE grafts
endothelialization
and thrombin
cell (SMC)
has developed
and in viva following
Thoraco-abdominal
capsules
muscle
Our laboratory
in
proliferative
may be useful
affects
following
with
release
of that protein. Keywords:
Tissue
engineering,
vascular
grafts,
FGF, EC, SMC, heparin
Received 12 December 1994; accepted 17 February 1995
many investigators to pursue strategies aimed at optimizing the biological interaction with the synthetic polymer rather than to minimize all biological interaction. Thus, the term ‘biocompatible’ has had a subtle change in its definition from a relative lack of biological response to a relatively desirable set of biological responses. The implanted polymer instantaneously affects the surrounding biological milieu, resulting in alterations in protein structure, platelet activation, inflammatory response, etc. By the same token, these biological responses necessarily alter the surface and to some extent bulk properties of the polymer and thus a continuous dynamic is achieved by which both the biological milieu and the polymer each alter the behaviour of the other. This results in the establishment of a novel ‘prosthesis-tissue complex’ whose properties differ from either component alone. Recent research has led to some understanding of how specific chemical and/or biomechanical parameters of the polymer may alter a variety of biological responses including the generation of growth factors by locally recruited
Research aimed at the development of more clinically efficacious small-diameter vascular grafts has been profoundly influenced by two major conceptual advances in our understanding of arterial wall biology. When Voorhees et a1.l reported the first successful application of a synthetic vascular graft, in this case made of Vinyon N, the arterial wall was considered to be a simple conduit through which blood passed. Since that time, however, an extraordinary expansion of our knowledge of arterial wall biology has documented an exceedingly complex set of metabolic activities by arterial wall cells. This advance has led to a more sophisticated approach to research in arterial replacement materials. A concomitant advance has occurred in the field of biomaterials. The long sought after biologically inert material has not as yet been developed, and the realization of the inherent ‘biointeractivity’ of all implanted polymers has led *Presented at the Biomedical Engineering October 1994. Correspondence to Dr H.P. Greisler.
Society
Meeting,
329
Biomaterials
1996. Vol. 17 No. 3
330
Biointeractive
polymers
cells, resulting in dramatic alterations in the degree of smooth muscle cell, myofibroblast and endothelial cell recruitment to the implanted bloodcontacting biomaterial. We and others have pursued a series of investigations demonstrating the dramatic difference in mesenchymal tissue incorporation induced by different polymeric materials and documenting the role of locally secreted growth factors in response to these implants as mediators of these different healing responses. For example, the implantation of lactide-glycolide copolymerit biomaterials induces local macrophages to synthesize, among other proteins, basic fibroblast growth factor (FGF-2), resulting in a significantly thicker and more cellular inner capsule as compared to either Dacron or expanded polytetrafluoroethylene (ePTFE) materials. Using this observation, we have pursued studies of methods of applying exogenous fibroblast growth factor to ePTFE to induce similar mesenchymal tissue ingrowth. When vascular prostheses are constructed of either polyglycolic acid (PGA) or of Dacron to nearly identical weave characteristics, wall thickness, including porosity and elastic modulus, the tissue ingrowth following implantation of these two materials differs greatly. Serial explants at time intervals ranging from 2 weeks to 1 year following implantation into rabbit i&arena1 aortas (48 PGA and 21 Dacron) were studied by gross and histological techniques”,“. The PGA material was resorbed between 4 weeks and 3 months following implantation. No rabbit displayed evidence of haemorrhage, perigraft haematoma, false aneurysm or graft infection. The PGA explants displayed a 10% incidence of aneurysmal dilatation. Inner capsule thickening increased significantly in the PGA group from 2 weeks until 2 months, at which time it stabilized at 3.17 times greater than that reached in the Dacron control group. Differences in tissue thickness resulted from differences in cell numbers and extracellular matrix content. Histologically, by 2 weeks macrophages and foreign body giant cells infiltrated the PGA interstices and inner capsules, and intracellular PGA inclusions were found as evidence for phagocytosis. By contrast, these inflammatory cells abutted the Dacron material but without evidence of intracytoplasmic Within 2 weeks of the appearance of inclusions. phagocytosis of PGA by macrophages, the inner capsules dramatically thickened with numerous circumferentially and longitudinally oriented layers of myofibroblasts amidst densely packed collagen, as shown in the 4 week PG910 explant represented in Figure lb and controlled with the 4 week Dacron explant shown in Figure la. These cells stained positively with an anti-sc-actin antibody and were covered along the blood-contacting surface by a confluent layer of Factor VIII positive endothelial cells, which by transmission electron microscopy were demonstrated to contain Weibel-Palade bodies. By contrast, at this same 4 week time point the inner capsule within Dacron prostheses remained a fibrin coagulum with minimal mesenchymal cell incorporations. By 3 months the PGA was fully resorbed, the macrophage population gone, and the neointima and flow surface changed little from 3 to 12 months.
and tissue
engineered
blood
vessels:
HP.
Greisler
et al.
inflammatory
Biomaterials 1996. Vol. 17 No. 3
Figure 1 Top: mid-portion of a Dacron specimen explanted after 1 month showing inner capsule (IC) composed of fibrin coagulum and minimal cellular repopulation (haematoxylineosin, original magnification x74). Bottom: mid-portion of 1 month polyglycolic acid specimen showing greatly thickened IC of circumferentially and longitudinally orientated smooth muscle-like myofibroblasts beneath endothelial-like flow surface. Progressively more peripheral to the lumen are the prosthesis and the well-vascularized outer capsule (haematoxylin-eosin, original magnification x74). (Reproduced from Ref. 3, by permission of the American Medical Association.)
Slower resorption produced by an increase in the ratio of 1actide:glycolide rings resulted in a similar but more slowly induced tissue ingrowth. This altered chemistry was used in production of polydioxanone (PDS) grafts. Twenty-eight PDS prostheses were implanted into rabbit aortas and harvested from 2 weeks to 1 year4. Among these explants, mild aneurysmal dilatation was found in l/28 and no stenoses or occlusions were encountered. The rate of tissue ingrowth paralleled the kinetics of macrophage mediated prosthetic resorption, with progressive inner capsule thickening ending after 3-6 months at 420 pm, a thickness not statistically different from the maximum of 480 pm found after 2 months in the PGA specimens. Capillarization of the inner capsules apparently resulting from a transinterstitial ingrowth from the surrounding tissue was found to communicate with the blood-contacting surface and was considered the source of the confluent endothelial layer along that surface (Figure 2). Following explantamechanical tion, studies demonstrated these regenerated tissue conduits to display arterial-like elasticity characteristics and resistance to fatigue or bursting at 6000 mmHg (-798MPa) peak systolic and 2000mmHg (-266 MPa) mean pressures. Freshly
Biointeractive
Figure 2
polymers
Mid-portion
and tissue engineered
blood vessels: HP. Greisler etal.
than those found within prostheses similarly woven of yarns containing 100% PG910. Support for the observation that inner capsule tissue ingrowth within bioresorbable prostheses results from a transinterstitial migration came from analyses of compound prostheses in which a central PG910 segment (10 mm long) was placed between proximal and distal Dacron segments (10 mm) and implanted into rabbit i&arena1 aortas. These grafts were explanted in triplicate. The inner capsule thickness within the PG910 segments increased from 2 weeks to 2 months and was significantly thicker than either Dacron segment at both 1 and 2 months (P < 0.004 and P < 0.0001,respectively). At these time points the inner capsules within the Dacron segments remained devoid of mesenchymal tissue beyond 2 mm of pannus ingrowth. The junctions between the inner capsules of the two materials were abrupt. Inner capsule thickening results not only from cell migration and extracellular matrix deposition but prominently from proliferation of ingrowing endothelial cells and myofibroblasts. Mitotic activity within inner capsules was studied using autoradiographic techniques to demonstrate incorporation of tritiated thymidine into newly synthesized DNA. PG910, PDS and Dacron prostheses were implanted into the infrarenal aortas of rabbits and specimens harvested from 2 weeks to 3 months following implantation. Autoradiographic analyses on crossadministration of following tritiated sections thymidine demonstrated a significant increase in mitotic index within the two bioresorbable groups, the rate of cell proliferation paralleling the course of prosthetic resorption (Table l)fi. PG910 explants showed a mitotic index of 28.3 + 23.2% 3 weeks after implantation, this proliferative activity progressively decreasing to 1.2 + 1.1% after 12 weeks. The more slowly resorbed PDS group demonstrated a more prolonged elevation of mitotic activity, 7.5 & 5.6% after 12 weeks. The Dacron group, by contrast, never exceeded proliferation rates greater than 1.2 & 0.9%. The location of the labelled proliferating cells was deep in the inner capsules in proximity to the macrophages and prosthetic material. In this zone the myofibroblasts demonstrated the synthetic ultrastructural phenotype commonly displayed by actively cycling cells”. Similarly, collagen content within the inner capsules parallels the kinetics of prosthetic resorption7. Collagen content measured by hydroxyproline calorimetry within the inner capsules 1 month following implantation of PG910 prostheses measured
of PDS specimen at 2 months showing
capillary invading inner capsule and communicating with luminal surface. Endothelialized luminal surface of specimen appears to be continuous with capillary wall (haematoxylineosin, original magnification x268). (Reproduced from Ref. 4, by permission of American Medical Association.)
explanted tissues were perfused and perfusates assayed for 6-keto-PGF,,/TxB, ratios before and after the administration of arachidonic acid. Results from tissues explanted at 6 and 12 months (1.62 i 0.70) did not statistically differ from control rabbit aortas (1.85
+I 0.61).
The construction of woven prostheses made from compound yarns containing both PGA or polyglactin 910 (PG910) plus Dacron components results in a significant diminution in tissue ingrowth and inner capsule cellularity”. The inner capsules within prostheses constructed of yarns containing 80% PG910 and 20% Dacron were statistically significantly thinner Table 1
Mitotic
331
index (%) PG910
PDS
Dacrona
2 3
1.42 f 0.59 28.34 f 23.21
No IC
No IC -
4 12 52
7.58 f 2.02* 1.17 f 1.05” 0.72 f 0.98””
7.50 * 2.66 7.50 i 5.59 1 .oo i 0.22****
No IC
Implant
aDue
to the
the other ‘P
duration
(weeks)
relativehypocellularlty
groups
< 0 01 versus
are
lmposslble
12 week
Dacron;
of the inner However, “P
c
the
capsules
(ICs)
hypocellularity
0 01 versus
4 week
prior itself PG910.
to 12 weeks
in the Dacron
is due largely *“P
group,
It 0.3v** 1.22 l 0.90*** 1.18
statistlcal
to the lack of mltot!c
<: 0 05 versus
4 week
PDS,
““P
activity
comparisons
in mltotlc
index
between
Dacron
and
in thts group
c 0 05 versus
4 week
PDS.
Biomaterials
1996.
Vol.
17
No. 3
Biointeractive
332
35.5 5 9.9 compared
polymers
pg (mg collagen))’ rng-’ dry weight to 25.6 i 5.0 in the PDS group and 12.0 * 0.9 in the Dacron group (P < 0.02 or P < 0.01 comparing PC910 or PDS to Dacron, respectively). The collagen content within the normal rabbit aorta measured 22.5 f 1.2 pg mgg. Based upon the temporal and spatial correlation between proliferating cells and macrophages, we that macrophage-biomaterial interhypothesized actions may yield a differential activation of the macrophage, inducing a differential secretion of bioactive factors either stimulatory or inhibitory to myofibroblast, smooth muscle cell and endothelial cell proliferation. We further hypothesized that this secretion of growth modulators played a major role in the initiation of the extensive mesenchymal cell ingrowth seen following implantation of lactideglycolide copolymeric bioresorbable prostheses. To test these hypotheses, unstimulated peritoneal macrophages harvested from New Zealand White rabbits and identified by immunoperoxidase staining using the rabbit macrophage specific RAM 11 antibody were cultured in vitro. To the culture media were added particles of either PG910, Dacron or neither material. Conditioned media were assayed for mitogenic activity using growth assays of quiescent BALB/c 3T3 cells, rabbit aortic smooth muscle cells and LE-II murine capillary lung endothelial cells. Macrophages exposed in vitro to PG910 released into their culture media substances which induced significantly more DNA synthesis in BALB/c 3T3 cells (1.6-3.3 times) as compared to macrophages exposed to Dacron or to neither biomaterial. Similar results were found in bioassays using smooth muscle cells and endothelial cells (Figure 3)‘. The greatest mitogenic activity within the conditioned media of macrophages exposed to PG910 was encountered during weeks 3-5 of culture, a time corresponding to visually apparent PG910 phagocytosis by the cultured cells.
MACROPHAGE
CONDITIONED
RABBIT
SMC
MEDIA
STUDY
BIOASSAY
Figure 3 Tritiated thymidine incorporation into normal rabbit aortic smooth muscle cells exposed to conditioned media collected weekly from normal rabbit macrophages cultured in the presence of PG910, Dacron or neither biomaterial (mean fs.d., P < 0.05 for PG910 versus control weeks 1-5, and Dacron versus control weeks 1 and 2). Biomaterials
1996, Vol. 17 No. 3
and tissue
engineered
blood
WCKOPHAGE
vessels:
CONDIIIONED RABBIT
NE,‘TRAI
SMC l7lNG
HP.
MEL’IA
Greisler
ef al.
STUDY
BIOASSAY AhTImbiir
Bt,
Figure 4 New Zealand White rabbit aortic smooth muscle cell bioassay using conditioned media from macrophages harvested from rabbits and exposed for l-7 weeks to either no biomaterial, polyglactin 910 or Dacron. The solid bars represent the results in the absence of the neutralizing anti-basic FGF antibody and the patched bars represent the results following the pre-incubation of conditioned media with a concentration of neutralizing anti-basic FGF antibody equal to five times the NDSO (mean f sd.).
The smooth muscle cell bioassays were repeated following incubation of the conditioned media with a neutralizing anti-basic FGF antibody (Upstate Biotechnology, Lake Placid, NY, USA). The two- to three-fold increase in smooth muscle cell proliferation induced by macrophage exposure to PG910 was diminished by 50-80s (Figure 4). A similar reduction in mitogenicity of conditioned media of macrophages exposed to Dacron was induced by this neutralizing antibody. By contrast, no change in mitogenicity of media of macrophages in the absence of biomaterial was found. These results corresponded to Western blot analyses showing immunoreactivity with an antibody against FGF-2 in the conditioned media of macrophages exposed to either biomaterial but no immunoreactivity of the media of macrophages in the absence of biomaterials. Parallel experiments evaluating the presence of transforming growth factor-a (TGF-/I) in conditioned media suggested that macrophages exposed to Dacron released into the media significantly greater amounts of bioactive TGF-P as compared to macrophages not exposed to biomaterialsg,lO. This may be interpreted either as a greater synthesis and/or secretion of active TGF-P, [as shown by neutralization of activity by preincubation with a neutralizing anti-TGF-/?, antibody), or conversely, as a greater degree of activation of latent TGF-P, the degree of secretion of which may or may not have been altered by interactions between the macrophages and Dacron. The studies described above suggest that different biomaterials may induce dramatically different degrees of smooth muscle cell, myofibroblast and endothelial cell ingrowth via a transinterstitial migration with a transient proliferative response, and that this is initiated at least in part by production of growth factors by activated macrophages. We next reasoned
Biointeractive
polymers
and tissue engineered
blood vessels:
that the application of specific growth factors to biomaterials may induce a similar response. In early studies published in 198611,‘2, we affixed FGF-1 (acidic FGF) to Dacron and to polydioxanone surfaces by sequential application of fibronectin followed by heparin (via its fibronectin affinity), FGF-1 (via its heparin affinity) and finally a second heparin layer because of the ability of heparin to protect FGF from proteolytic degradation as well as its synergistic activity with the growth factor. The retention of lz51FGF-1 by the preparation prosthesis following implantation into the rabbit aorta was quantitated. Following 1 week in circulation, grafts were explanted and FGF-1 eluted using salt solutions, and the FGF-1 was shown to have retained its molecular integrity as determined by polyacrylamide gel electrophoresis. This eluted FGF was shown to have retained its mitogenic activity in culture, inducing the anticipated DNA synthesis when added to quiescent murine capillary lung endothelial cells. However, using this FGF application technique, no enhancement of in vivo spontaneous endothelial cell ingrowth or proliferative activity of seeded endothelial cells was documented. Further in vitro studies led us to conclude that FGF-1 bound to immobilized heparin did not possess mitogenic activity until the bond either between the FGF-1 and heparin or between the heparin and fibronectin was broken. The ability of a variety of FGF delivery systems to promote endothelial cell growth was studied in a cell culture model. Surfaces pretreated with fibronectinl heparin/FGF-llheparin did not induce growth of human umbilical vein endothelial cells or canine jugular vein endothelial cells when soluble FGF-1 was excluded from the media. Using the same assay system, we screened FGF affixation techniques and found the binding of FGF-1 to any immobilized substrate rendered FGF-1 devoid of endothelial cell mitogenic activity. However, utilizing the same assay we developed a method currently employed in our laboratory in which FGF-1 and heparin are placed into suspension within a fibrinogen/thrombin matrix (fibrin glue)13. In vivo release kinetics were quantitated using fibrin glue suspensions containing 1251-FGF-1 and heparin impregnated into ePTFE grafts implanted into rabbit infrarenal aortas. After 7 and 30 days in circulation, 13.4 +6.9% and 3.8 f l.l%, respectively, of the applied 1251-FGF-1 remained on the prostheses. When calculated as percent changelmin change, the rate of loss during the initial 1 h averaged -24.1 A%lA min. After this initial hour the rate of loss from 60 min to 30 days averaged -0.03 A%lA min. The distribution of the 1251-FGF-1 across the wall of the prostheses was linear, with equal radioactivity found in each successive 1/18th segment of the 600 pm thickness of graft wall, documenting the application technique capable of distributing the FGF-1 along the inner as well as outer graft surfaces and throughout the internodal spaces. Utilizing this impregnation technique, we evaluated the effect of this fibrin glue/heparin/FGF-1 preparation on graft healing, endothelialization and endothelial cell proliferation in a bilateral canine aorto-iliac
HP.
Greisler
et al.
333
bypass graft model13. ePTFE grafts, 60 pm internodal distance, treated with fibrin glue/heparin/FGF-1 (Group I) were implanted contralateral to either ePTFE grafts treated with fibrin glue/heparin but devoid of FGF-1 (Group II) or untreated ePTFE grafts (Group III). Grafts were explanted after 7 or 28 days. At 28 days every Group I explant displayed a totally confluent Factor VIII positive endothelialized surface and extensive capillarization through the internodal spaces of the graft wall from the surrounding tissue. By contrast, no specimen of either Group II or Group III displayed a confluent endothelialized surface, and only occasional small islands of cellularity were observed interspersed with areas of fibrin coagulum populating the inner capsules. En face autoradiographic analysis of tritiated thymidine incorporation into newly synthesized DNA (Table 2) documented a significant increase in endothelial cell mitotic activity in the 28 day explants pretreated with fibrin glue containing heparin and FGF-1 (P < 0.05). Further studies were performed to evaluate the potential of this FGF-1 application technique to induce endothelialization over longer length grafts as well as the potential for this system to induce myointimal hyperplasia at later time points14. Thoraco-abdominal aortic implants (30 cm length x 8 mm i.d.) of 60 pm internodal distance ePTFE were again divided into the three groups described above. Grafts were explanted after 10, 30 or 140 days. All 26 explants were patent and none showed evidence of thrombus formation, stenosis or dilatation. No gross or histological evidence of anastomotic intimal hyperplasia was seen. Histologically, the untreated grafts were lined solely with fibrin coagulum through 30 days, whereas at 20 weeks small islands of pseudo-intima containing several layers of smooth muscle cells covered portions of the explants interspersed with areas of thrombus. The specimens pretreated with FGF-1 and explanted after 10 days showed an extensive mesenchymal tissue ingrowth into the body of the graft, with smooth muscle cells and capillaries extending 80% of the distance from outer to inner graft surface. By 30 days the entire graft was extensively infiltrated with abundant capillarization, and the blood-contacting surface was nearly 100% confluent and positive for the presence of Factor VIII by immunoperoxidase staining. This endothelialized neointima remained throughout the entire 30 cm graft length at 140 days, again with extensive capillarization throughout the graft wall (Figure 5A and B). The source of the capillary ingrowth was apparently a migration from the surrounding tissue. Along the outer surface, capillary
Table 2
Number of labelled cells per high-power field (x40)
Group I (FGa/heparin/FGF-1) Group II (FG/heparin) Group Ill (untreated) * FG : fibrin
7 days
28 days
3.01 * 1.95 1.72 ?c 1 .Ol 1.45 It 0.49
22.18 zt 12.87’ 2.42 & 1.65’ 1.09 f o.55c
glue.
bP
< 0.02
versus
Group
‘P
< 0.05
verws
Group
I (7 I (28
days). days)
Biomaterials 1996, Vol. 17 No. 3
334
Biointeractive
polymers
A
and tissue
engineered
blood
vessels:
HP.
Greisler et al.
B
Figure 5 A, Photomicrograph of an FG/FGF-l-treated graft explanted after 20 weeks showing a more developed inner capsule consisting of numerous layers of longitudinally orientated myofibroblasts and collagen and covered by an endothelial cell monolayer (haematoxylin-eosin. original magnification x724). 6, Photomicrograph of the same FG/FGF-l-treated graft showing abundant capillarization of the graft wall and two mitotic figures (arrows) (haematoxylin-eosin, original magnification x724)
infiltration of the internodal spaces (Figure 6A) could be seen at all time points in grafts pretreated with the FGF-1. As early as 10 days following implantation, capillaries could he seen extending through the graft wall and reaching the blood-contacting surface of the graft (Fi,our’r h‘B and C), with continuity of the endothelial cells between ingrowing capillaries and the blood-coIitac:ting surface of the implanted graft. This abundant mesenchymal tissue and capillary infiltration was accompanied by a transient FGF-1 induced increase in cell proliferation. Cross-sectional autoradiography (Fi,nure 7) showed at 30 days the effect of FGF-1 in inducing a proliferative response, with 9.6 i 4.4 of the cells picking up the tritiated following during the 10 h period thymidine thymidine administration. At 20 weeks the mitotic index had decreased in all groups, measuring less than 1% with no difference among the groups. The inner capsule thickness among 20 week explants, however, revealed a greater degree of thickening in the grafts pretreated with the FGF-1 (139 i 178 pm). No significant difference was seen comparing the peri-anastomotic and more central zones.
IJsing reverse transcription polymerase chain reaction techniques, we evaluated the expression by inner capsular cells of mRNA for FGF-1, FGF-2, TGFType I (flg) to ascertain B, and the FGF receptor whether the presence of exogenous FGF-1 might induce a secondary upregulation of other growth factor genes which might over time result in intimal hvperplasia’4. No difference was seen in the level of expression of these growth factors when comparing results between the three groups of grafts. Northern blot analysis for platelet-derived growth factor (PDGF) A and PDGF B chain message similarly showed no apparent difference in the level of expression as a function of either time or graft treatment method. Further in vifro studies in our laboratory have been designed to modify the FGF-1 : heparin ratio so as to solectivel! induce endothelial cell proliferation without a concomitant increase in smooth muscle cell exists proliferation”. A complex relationship between FGF-1 and heparin as concerns smooth muscle cell proliferation. Heparin synergizes with FGF-1 in stimulating the proliferative response of smooth muscle cells. whereas heparin in the absence
Biointeractive
polymers
and tissue
enoineered
blood
vessels:
HP.
Greisler eta/.
335
C Figure 6 A, Photomicrograph of an FG/FGF-l-treated graft implanted after 20 weeks showing the abluminal portion of the graft wall and adjacent outer capsule. Numerous capillaries, C, can be seen with some transversing the tissue-material interface and entering the graft’s internodal spaces (haematoxylin-eosin, original magnification x825). B, Photomicrograph of the same FG/FGF-l-treated graft as that shown in figure5A showing abundant capillarization, C, filled with red blood cells in the graft wall and showing a mitotic figure (arrow) (haematoxylin-eosin, original magnification x825). C, Photomicrograph of an FGIFGF-l-treated graft implanted after 30 days showing the luminal portion of the graft wall with a capillary, C, extending to the luminal surface and with endothelial cells (arrow) lining both the capillary and the luminal surface (haematoxylin-eosin, original magnification x825)
of FGF-1 inhibits smooth muscle cell proliferation. A synergism between FGF-1 and heparin in stimulation of endothelial cell proliferation occurs, but no heparin-induced inhibition of endothelial cell proliferation has been reproducibly documented. We thus hypothesized that there might exist a ratio of FGF-1 :heparin that selectively supports the growth of endothelial cells as compared to smooth muscle cells. In a series of in vitroanalyses, we evaluated the growth of both rabbit and canine arterial smooth muscle cells in response to either soluble FGF-1 and heparin or to FGF-1 and heparin within suspensions of fibrin glue, upon which the smooth muscle cells were cultured. Smooth muscle cell proliferation was significantly increased when either 5 or 50 Uml-’ of heparin (+85% and +66%, respectively, P < 0.0001 for both) was added to 10 ng ml-l of FGF-1. However, smooth muscle cell proliferation was significantly decreased from control (-IL?%, P= 0.014) by the addition of 500 U ml-l of heparin. Concomitant
studies on human umbilical vein endothelial cell proliferation showed a significant increase by the addition of 5, 50 or 500 U ml-’ heparin (+68%, +99% and +lOS%, respectively, P < 0.0001 for all). No significant inhibition of endothelial cell proliferation was found at even the highest, 500 UmlK’, heparin concentration studied. The effect of this FGF1: heparin ratio on selectively inducing endothelial cell proliferation without smooth muscle cell proliferation in vivois currently being evaluated. From these studies we conclude that different polymers, by their different biochemical characteristics and/or biomechanical properties, are capable of inducing dramatically different degrees of mesenchymal tissue ingrowth. The mechanism in part appears to be a differential for this difference activation by the polymers of macrophages with which they interact, resulting in a differential production of growth factors which may either stimulate or inhibit endothelial cell, smooth muscle cell and myofibroblast Biomaterials
1996,
Vol. 17 No. 3
Biointeractive
336 CROSS
SECTIONAL TRITIATED
polymers
and tissue
THYMIDINE
2 3
4
5
6
7 140
DA”;
Figure 7 Mitotic index expressed as number of labelled cells per 100 cells within the inner capsules (mean + s.e.m.).
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proliferation and migration. A similar alteration in ingrowth of these cells may be induced by application of exogenous growth factors when impregnated into prosthetic materials in a biologically active delivery system. The ideal polymers and the ideal growth factors as well as the ideal delivery systems have certainly not yet been determined. We contend, however, that by use of these techniques the biological response to implanted biomaterials may be selectively regulated so as to result in the regeneration of functioning arterial conduits following implantation of partially bioresorbable or either bioresorbable, relatively inert but non-resorbable biomaterials.
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ACKNOWLEDGEMENTS Supported in part by grants from the National Institutes of Health, numbers ROl HL29268 and ROl HL41272. The authors wish to acknowledge Mr Richard Dewitt for the preparation of the photomicrographs, MS Jacqueline Garfield for the electron microscopy preparation and Peter I. Kim for his assistance in the electron microscopic analyses.
3
vessels:
HP.
Greisler
et al.
Voorhees AB, Jaretzki A, Blakemore AH. The use of tubes constructed from Vinyon N cloth in bridging arterial defects. Ann Surg 1952; 135: 332. Greisler HP. Arterial regeneration over absorbable prostheses. Arch Surg 1982; 177: 1425-1431. Greisler HP, Kim DU, Price JB, Voorhees AB. Arterial regenerative activity after prosthetic implantation. Arch Surg 1985; 120: 315-323. Greisler HP, Ellingher J, Schwartz TH, Golan J, Raymond RM, Kim DU. Arterial regeneration over polydioxanone prostheses in the rabbit. Arch Surg 1987; 122: 715-721. Greisler HP, Schwartz TH, Ellinger J, Kim DU. Dacron inhibition of arterial regenerative activity. 1 Vast Surg 1986; 3: 747-756. Greisler HP, Petsikas D, Lam TM, Pate1 N, Ellinger J, Cabusao E, Tattersall CW, Kim DU. Kinetics of cell proliferation as a function of vascular graft material. J Biomed Mater Res 1993; 27: 955-961. Cabusao EB, Lam TM, Ellinger J, Greisler HP. Kinetics of collagen deposition within bioresorbable and nonresorbable vascular prostheses. Tram Am Sot Artif Organs (ASAIO) 1991; 20: 113. Greisler HP, Ellinger J, Henderson SC, Shaheen AM, Burgess WH, Lam TM. The effects of an atherogenic diet on macrophage/biomaterial ineractions. J Vast Surg 1991; 14: 10-23. Petsikas D, Cziperle DJ, Lam TM, Murchan P, Henderson SC, Greisler HP. Dacron-induced TGF-8 release from macrophages: effects on graft healing. Surg Forum 1991; 42: 326-328. Greisler HP, Petsikas D, Cziperle DJ, Murchan PM, Henderson SC, Lam TM. Dacron stimulation of macrophage TGF-P release. Cardiovasc Surg 1994; (in press). Greisler HP, Klosak JJ, Dennis JW, Ellinger J, Kim DU, Burgess WH, Maciag T. Endothelial cell growth factor (ECGF) attachment to biomaterials. ASAIO Tram 1986;
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blood
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Greisler HP, Klosak JJ, Dennis JW, Karesh SM, Ellinger J, Kim DU. Biomaterial pretreatment with ECGF to augment endothelial cell proliferation. J Vast Surg 1987; 2: 393402. Greisler HP, Cziperle DJ, Petsikas D, Murchan PM, Appelgren EO, Drohan W, Burgess WH, Kim DU. Enhanced endothelialization of expanded PTFE grafts by heparin binding growth factor-type I (HBGF-1). Surgery 1992; 112: 244-255. Gray JL, Kang SS, Zenni GC et al. FGF-1 affixation stimulates ePTFE endothelialization without intimal hyperplasia. J Surg Res 1994; 57: 596-612. Kang SS, Ren D, Gosselin C, Greisler HP. Smooth muscle cell inhibition with endothelial cell stimulation by growth regulators within a fibrin glue delivery vehicle. Surg Forum 1994; 45: 355-358.