Quantitative assessment of the tissue response to implanted biomaterials

assessment of the tissue responseto implantedbiomaterials D, Geoffrey Vim, John A. Hunt and David F. Williams hstifute of Medical and Dental ~ioengineering, University of Liverpool, PO Box f4 7, Liverpool fRecewed 25 August f990; revised 20 November 7990; accepted 20 December 199Oj

L69 3BX. UK

The tissue response to a small number of polymeric biomaterials wes studied using monoclonal antibodies specific for certain inflammatory cell types, to develop a reliable and accurate method for the quantitative evaluation of biocompetibility. The sites of antibody binding were identifed using an avidin-biotin staining procedure and the sections evaluated using a computer-aided image analysis system. The staining technique successfully demonstrated both polymorphonuclear leucocytes and macrophagas in tissue samples containing polymeric biomaterials. The image analysis system facilitated the measurement of up to 30 cell-related pa~meters and allowed a large number of cells to be analysed. Keywords: image analysis, cell-material

interactions, bjocompaiibjij~

The concept of biocompatibility is based on the interactions between a material and a biological environment. The failure of a biomateriai, in a clinical situation, to display good bi~ompatibility is often revealed by a breakdown in the desired material properties or an unsatisfactory biological response. The most important aspect of biocompatibility, for the performance of the material. is the local tissue response, as this usually provides a clinical indication of a biocompatibility deficiency. Analysis of the local tissue response to a biomaterial has long been recognized to play an important role in biocompatibility testing. The normal wound-healing response is a dynamic phenomenon, in which cells and their products interact to repair damaged tissue. If an implant is present in the tissue, this sequence of events is disrupted to varying degrees, resulting in a visible change in tissue morphology. Many types of cell are involved in normal wound healing, including macrophages and polymorphonuclear leucocytes (PMNs). Wound healing in the presence of a biomaterial may induce a more complex reaction involving lymphoid and myeloid cells. Traditional staining techniques, such as haematoxylin/ eosin, van Giesen, or Periodic Acid-Schiff, are often used in the classification of inflammato~ cell types. These cells, which include macrophages, PMNs, T-lymphoc~es and Blymphocytes, can then be identified by morphological characteristics. In the assessment of the local tissue response to implanted biomateriais, the observed distribution of these cells may be used to provide a qualitative description of the reaction. Attempts to provide a more quantitative Correspondence to Professor D.F. Williams 0 1991

Butterworth-Heinemann

assessment have involved cell counting, using morphological criteria, and the allocation of a grading of the response’, usually on a scale of 1 to 5. This method of evaluation relies on the subjective assessment by the operator, leading to errors such as misidentification of cell types or miscounting of cells. In an attempt to overcome some of these problems and to quantify accurately the complex interactions between ceils and the surrounding tissue, a number of staining methods have been investigated and used in conjunction with a computer-aided image analysis system.

MATERIALS

AND

METHODS

To study the biocompatibility of polymeric biomaterials, a series of polymers were implanted bilaterally into the dorsolumbar musculature of black and white hooded Lister rats. Two animals per material were used for each time period, with each rat implanted with two pieces of the same material. The materials used in this study comprised poly(glycolic acid) (PGA), (polylactic acid) (PLA), 50% PLA/ 50% PGA copolymer (Medisorb, DuPont, USA) and BiomeP). After periods of up to 3 months, the rats were killed by cervical dislocation, and the tissue surrounding the implant carefully removed. The tissue, with the implant in situ, was frozen using isopentane and dry ice, and sectioned at 7 pm using a cryostat microtome. Each tissue block was trimmed down to the edge of the implant and serial sections taken for staining. Two staining methods were used. The first of these is a rapid enzymic technique to detect chloroacetate esterase. This enzyme is found almost exclusively in PMNs and mast

Ltd. 0142-9612/91/080731-06 Biomarerials

799 1. Vol 12 Ocrober

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Tissoe response to biomaterials: D.G. Vince et al

cells. These cells can be detected by incubating tissue sections with naphthol AS-D chloroacetate in the presence of freshly formed diazonium salt. Enzymic hydrolysis of ester linkages liberates free naphthol compounds which couple with the diazonium salt, forming highly coloured deposits at sites of enzyme activity. This method can be carried out quickly and conveniently using a commercial test kit (Sigma, Code 91 -A). Minor modifications were made to the method sheet provided. Fixation of the sections before staining was by using a citrate-acetone-methanol solution, followed by thorough rinsing. The citrate-acetone-formaldehyde fixative recommended for use with the kit was found to suppress staining of PMNs. A developing solution was prepared as follows: sodium nitrate solution (50~1), fast red violet LB base solution (50 PI), distilled water at 37°C (4 ml), trizmal buffer pH 6.3 (250~1) and naphthol AS-D chloroacetate solution (50~1). The sodium nitrate solution and fast red violet LB base solution were mixed gently by inversion and allowed to stand for 2 min. The distilled water, trizmal buffer and naphthol AS-D chloroacetate solution were then added to the mixture. This was applied directly to the slides and incubated for 60 min at 37”C, protected from the light. Finally, the slides were rinsed in distilled water for 2 min. The slides were than coverslipped using aqueous mounting media before undergoing image analysis. If morphological examination was required, haematoxylin counter staining was performed before mounting (Figure 1). Identification of the other inflammatory cells was performed using an immunohistochemical staining method. The avidin-biotin technique represents one of the most recent developments in immunoperoxidase staining and is based on the ability of the egg-white glycoprotein avidin to bind non-immunologically four molecules of the vitamin biotin. Three primary reagents are used in this technique. The first is a primary antibody specific for the antigen to be localized. Murine monoclonal antibodies towards specific rat surface antigens were obtained. The monoclonals selected were as follows. ED2. This recognizes a membrane antigen present predominantly on tissue macrophages of the rat. Monocytes, dendritic cells, lymphocytes and granulocytes are negative for ED2. CD8 type. This recognizes a determinant on the majority of thymocytes (90-959/o), a subset of peripheral T-cells, and

the majority of NK-cells. The antigen recognized is a complex of surface glycoproteins and is the rat homologue of the human CD8 antigen and the mouse Ly2. The antibody labels a T-subset which mediates suppression of antibody formation and the cytotoxic cell precursor. IL2 receptor. This recognizes a glycoprotein found on activated rat T-cells but not resting lymphocytes. L-CA. This is a mouse monoclonal antibody to a subfraction of rat B-cell leucocyte common antigen (L-CA). This monoclonal binds only to B-cells. W3/13 HLK This displays specificity towards PMN, all thymocytes and T-lymphocytes and haemopoietic stem cells, but not B-lymphocytes. The ED2, CD8 type and the IL2 receptor monoclonal antibodies were obtained from Serotec and used at 1: 100 dilution, with the exception of CD8 type which was used at 1:40. These dilutions were decided on the basis of titration data obtained from the suppliers. The L-CA and the W3/13 HLK were purchased from Seralab; these two antibodies were supplied as supernatant and therefore used neat (Table 7). The second reagent used is antimouse polyclonal antibody (Dako, Code E413) and is covalently linked to one molecule of biotin. The third reagent is a complex of peroxidase conjugated biotin and avidin (Dako, Code K355). The free sites on the avidin molecule allow binding to the biotin on the second antibody. The peroxidase enzyme, and therefore the original antigen, are identified with an appropriate chromogen (Figure 2). The technique was performed as outlined below. Sections were cut at 7 ,um and fixed in acetone for 10 min before air drying. To destroy any endogenous peroxidase activity, the slides were incubated for 20 min in 0.6% hydrogen peroxide in methanol. Following rinsing in distilled water, the slides were placed in a bath of phosphate buffered saline (PBS) for 10 min. The sections were then dried by inverting on to paper towels and incubated for 25 min with rabbit serum diluted I:5 with PBS. This stage of the staining procedure blocks any rodent cross-reactivity antigenic sites. It is therefore important that immediately after this stage the slides are not rinsed but dried again by inversion on to paper towels. The serial sections were then incubated for 90 min with the monoclonal antibodies listed in Table 1, before rinsing in PBS for 5 min and drying by inversion. The rabbit antimouse polyclonal antibody was diluted to 1 : 100 and two drops of rat serum added to remove any antibodies which may cross-react with rat antigens. This solution was applied to the sections and incubated

Table 1 Antigen

Specificity

Form

Dilution

ED2

Macrophage T-lymphocytes (cytotoxic/ suppressor cells) Activated T-lymphocytes B-lymphocytes

Ascities Ascities

l:lOOofZ-lOmg/ml 1:40 of 3.39 mg/ml

Asclties

1: 100 of 3.80 mg/ml

Supematant

Neat at 5- 10 mg/ml

Supematant

Neat at 5-l 0 mg/ml

CD8 tvpe

Figure 1 Chloroacetate stained muscle tissue.

732

Biomaterials

esterase

stain showing mast cell in counter-

1991, Vol 12 October

Antigenic specificity and dilution of monoclonal antibodies

lnterleukln 2 receptors Leucocyte common antigen subfraction W3/13 HLK T-lymphocytes

Ttssue

with these methods.

l

ABC Method

Although

the tissue was expected the separations, cell types.

dase method

included

The avidin-biotin

serum reduced

in the positive

antibodies

use of 0.6%

the background

at

periods,

only

materials,

and

and

although reliable

the

PMNs

stated

were

not

time

present

in these

as a function

analysed

represent&on

of

the

are

(539

area, corresponding

to a defined

to Biomer produced

the maxrmum

cells) by 2 d. The cell number

in the tissue adjacent only 161

101

surface

the section.

sharply to 2 1 cells by 7 d. By 3 months,

14d, Diagrammatic

cell numbers

from the implant

dropped

the maximum

2

to

experiments.

of PMNs

no PMNs

In the

numbers

number

contrast,

numbers

of cells refers to those cells counted

The tissue response

Figure

large

in this section,

of distance

over a 20 frame within

in large

were detected

in sufficiently

analyses

and time. The number location

staining

All

these

In the data presented

1

the with

presented

x

thus increasing

image analysis system. although

e

Avldln

is

rat and rabbit

in the tissue sections,

present

A

An example

HzO,,

staining,

controls

to identify

of rat muscle

described.

(Figures 3 and 4). A few lymphocytes

Blotln

in

cell types were present

sections, Enzyme

et al.

immunoperoxi-

of sections

of the computer-aided

macrophages

KEY

Vince

of lymphocytes

indirect

gave clear staining

in Figure 3. The

sensitivity

the number

D.G.

the ability of this technique

with all the monoclonal shown

to b,omaterrals:

to be much lower than that found in

they were

as they demonstrate these

response

number

macrophages

(Figure 5). In

to the implant

macrophages

were

of cells found were

present

at 2 d, with

at 7 d (774

present

then

there were

in tissue,

cells). At with

the

avid/n-biotin-peroxldase

technique

for 40 min. The slides were applying

the avidin-biotin

rinsed in PBS for 5 min before complex,

incubated

for 30 min

and rinsed in PBS for 5 min. Sites of peroxidase identified (DAB).

by incubating

This was

with a solution

prepared

peroxide

was added.

incubated water

10 mg of DAB

in

60 p I of 3% hydrogen

This was applied

to the sections

and

for 15 min. The slides were then rinsed in distilled

for at least 2 min before

and mounting

dehydrating

through

for the stains comprised

samples

liver, blood and muscle tissue surrounding

copper/polymer

materials.

also performed,

in which

from whole

alcohol

in DPX.

Positive controls spleen,

of diaminobenzidine

by dissolving

15 ml of PBS. To 4 ml of this solution,

activity were

Ex viva positive rat lymphocytes

blood and stained

of

a range of

controls were

were

separated

using the panel of antibodies.

F/gore

3

Stavnng

of

macrophages

usv~g

avIdin-blot/n-peroxidase

techmoue

Histomorphometry The image

analysis

system

Loebl mini-Magiscan. Jenaval

photomicroscope

Hitachi

KP140

contains

to create

allow

that would

tissue

analyse

distance

of 640pm

from

analysed

area of 2.06

X

were

measured

from

implant

including

routines

executed,

analysis. the

implant,

cell number,

which

can be routines

routine

was

per slide, to a

providing

A number

using a

The Joyce-

these

A task

20 fields of view

1 O6 pm’.

under a Zeiss

captured

video camera.

menu-driven

task lists. When

fully automated

created

and the image

CCD monochrome

Loebl software linked

used in this study was a Joyce-

The slides were viewed

a total

of parameters

cell area, distance

and circularity.

RESULTS The

positive

lymphocyte

controls, separations,

involving

either

demonstrated

muscle successful

tissue

or

staining

Figure

4

Sta/nmg

of

PMNs

using

the

chloroacetate

esterase

stainmg

technique.

Biomaterrals

199 1, Vol

12 October

733

Tissue response f5 bioma?eria~s: D. G. Vince et al.

Figure 5 The distribution of PMNs Period: 1 = 2 d; 2 = 7 d: 3 = 14 d.

surrounding implants

of Biomeig.

Figure 8 The distribution of macruphages surrounding implants copolymer. Period: 1 = 2 6 2 = 7 d; 3 = 14 d; 4 = 3 months.

of

of 38 1 cells at 7 d. By 14 d, the number of macrophages had fallen to 319, and to 75 by 3 months (Figure 8). The PGA gave a similar response to the copolymer, with the number of macrophages reaching a peak of 963 cells at 7 d. The total number of macrophages decreased to 207 by day 14; data are not yet available for the 3 month time period (Figure 9). As expected, the number of PMNs reached a maximum at 2 d (690 cells), falling to only 55 cells by 7 d. By day 14, the number of PMNs had increased to 207 cells (Figure 70). The samples of PLA produced the greatest number

Figure 6 The distribution of macrophages surrounding implants of 8iomereS Period: 1 =2d;2= 7d;3= 14d;4=3months.

majority occurring within 40,um from the implant/tissue interface. By 3 months, the macrophages were evenly distributed throughout the section, with only 152 cells present in the tissue (Figure 6). The PMN response to the degradable PLA/PGA copolymer was quite different, with the number of cells increasing from 210 at 2 d to 427 at 14 d. As with the Biomer samples, no cells were found at the 3 month interval (Figure 7). The copolymer gave a similar response to Biomer with 327 macrophages present at 2 d, reaching a maximum

Figure 7 The distribution of PMNs surrounding impiants of copolymer. Period: 1 =2d;2= 7&3= 14d.

734

Biomaterials

199 1. Vol 12 October

Figure 9 The distribution of PMNs surrounding implants of PGA Per&d 1=2&2=7d;3= 14d.

Figure 10 The distribution of macrophages surrounding implants of PGA Period: 1 = 2 d; 2 = 7 d; 3 = 14 d.

Tissue

niques.

This

sensitive

method

has

(PAP)2. When

in conjunction

with

tool for the quantitative and wound

during

cells

a new extracellular

material

surface

sequence

physical Period:

1 =2d;2=

7d;3=

surrounding

implants

of PLA

14d;4=3months.

with

matrix.

the

wound

presence

may constitute

which

this

may

only be due to the the material

of irritation

response.

which

and relevant

to consider

mechanisms

of the normal wound-healing

could

process and may As the

require

implantation,

majority

of

it is convenient

biocompatibility

the presence

affect

Alternatively,

biomaterials

influence

is the

and static, and any deviation

degree

tissue

and

to the tissue may be small

of the implant.

surgical

other material

may occur between

may then

a significant

of a series of

If a foreign

tissue,

healing

in a different

is used of tissue

each

severely disrupt the normal wound-healing result

more

we have a

assessment consists

is inert, non-toxic

normal

technique

healing

of events. The stimulus

if the material from

to be

system,

interact

an interaction

and

et al.

healing.

of wound

which

Vince

such as peroxidase-

analysis

inflammation

D.G.

shown

this staining

the image

placed in the wound,

of macrophages

been

very powerful

produce

The distribution

also

antiperoxidase

The process

11

to biomatenals:

than other indirect techniques

events,

Figure

response

in the light of the

of an implanted

response,

and the

biomaterial

has on

this process. The tissue reaction towards response

to normal wound

cell type to appear

Biomer produced

healing. The PMNs

after surgery,

found close to the implant largely phagocytic,

with

surface.

although factor3.

the majority

platelet

activating

mately

1 d after which they are engulfed

Figure

12

The distribution

of PMNs

surrounding

implants

of PLA.

at 7 d, 14 d and sharply

Macrophages inflammation

macrophages majority implant.

present

of the After

2 d (488

number

occurring

those

(Figure

were

17).

within

out the tissue. the majority (Figure

within

macrophages

only 180

evenly

The

2 d (533

There

were

80pm

340

between

macrophages

distributed

maximum

from

were identified,

of cells occurring

pm. By 3 months,

and

cells).

in the tissue at the 7 d period, with the

cells

14 d, 346

the greatest 624

after

of

the

PMNs

of cells occurring

tissue

through-

had fallen to 250,

within

and

occurred

cells), and were evenly distributed By 7 d, the cell number

with

512

regulation Finally, debris,

with

64 pm of the implant

12).

macrophages

The sample

phagocytose

immunoenzymic

to

localize

technique

ments of the operator. primary

mouse

polyclonal

antirat

complex

used as an enzyme biotin is covalently avidin molecule

depends

on the

monoclonal

methods selection

individual technique

antibody,

secondary

and PMNs

present number

inflammatory

label. Biotinylation

occurring The

utilizes a

(HRP)

is a process whereby Free sites on the

from theavidin-biotin/HRPcomplex with

bind to

DAB used to identify

activity. The strong affinity of avidin for biotin sensitive

process than

direct

makes

the avidin-biotin

immunoenzymic

tech-

cellular

and

sequester

molecular

toxic materials.

a greater

tissue

response

of both macrophages the

time period,

the extent

may be difficult

the greatest

of any chronic

to judge, although

of macrophages

were

at

present,.

number of PM Ns occurred at day 2. to PLA had some similarities

with

the

maximum

number

on day 2, falling to no cells present response

the maximum

with

of PMNs

at 3 months.

gave the maximum

7 (43 cells at 64 pm). By 3 months,

performance

by

the

cells at day 2, although

vitro

response

and fibroblasts6.

on day 7. Without

number

count occurred

of

on day

the total cell number

had

value.

In a consecutively is

as

Secondly,

As with the Biomer,

number

fallen to its lowest

acute such

in the sections.

response

macrophage

preformed

peroxidase

to the antibody.

antibody,

which require-

monokines

of cells occurred

data for the 3 month

of the

a biotinylated

antibody,

and horseradish

and the mild biotinylation more

The

The avidin-biotin

attached

biotin on the secondary sites of enzyme

staining

antigens.

rabbit antimouse

avidin-biotin

method

and/or

The tissue response

used

of

of PGA invoked

14 d, the smallest

are many

appropriate

secretion

govern any ongoing inflammatory

that of the Biomer, be

roles in the inflamin controlling

of both T- and B-lymphocytes,

As expected,

There

major assist

than the Biomer, with a greater number

DISCUSSION

can

may

and also detoxify

maximum

until day 7

1 4, and by the release of metabolites5.

macrophages

were present

within

number

the

They

by their

interleukin

number

to 14 d. play several

response.

of these cells

The slower-moving

do not reach a maximum

matory of macrophages

3 months.

macrophages Period:

by macrophages.

number

and decrease 1 = 2 d; 2 = 7 d; 3 = 3 months.

is

source of

survive in viva for approxi-

This would explain the greatly reduced observed

of cells

The role of the PMNs

they are a significant

PMNs

a similar

were the first

running

project

of polymeric

investigating

materials,

the

the in

molecular

weights

of the PLA, PGAand

copolymerwere

specific

time periods.

were found to fall considerably

over a period materials release

These

of 14 d, when

appeared of particulate

to

breakdown

in distilled

hydrolyse,

debris.

noted, it can be assumed the

placed

measured

with

Since

no

a lowering

water.

over The

concomitant of pH was

that lactic acid and glycolic acid are

products.

As

these

t?/omatenals

two

199 1. Vol

compounds

12 October

are

735

Tissue response to biomaterials: 0.G. Vince et al.

soluble there will be no physical stimulation of the cells. This may explain the normal wound-healing response observed with these materials. The only material which did not follow this pattern was the copolymer, with the PMN rising at the 14 d interval before falling to undetectable levels at 3 months. This finding is as yet unexplained. This method of tissue evaluation has many advantages over the more commonly used staining/analysis procedures. One problem frequently encountered in the assessment of the tissue response to a biomaterial is that of cell jdentification. Morphological characteristics such as shape, size and nuclear area can be unreliable parameters, as these are dependant upon the state of activation and the motility of the cell. The immunostaining procedure overcomes this identification problem by using monoclonal antibodies that bind to surface antigens which are specific for the cell to be identified. The image analyser, when used in conjunction with the staining techniques, allows up to 30 cell-related parameters to be measured. Many of these, such as cell area, distribution and circularity are very difficult and timeconsuming to determine manually. Finally, as these cell measurements are fully automated, a large number of cells can be analysed without counting errors being introduced. This regime of histochemical staining and image analysis is a great improvement on the more routinely used tissue assessment techniques, in that small changes in the tissue response towards a material, or a group of materials, can be quantitatively assessed.

CONCLUSIONS The combination of histochemistry and image analysis greatly aids the quantitative assessment of this tissue response to biomaterials. This method of evaluation has many advantages over more commonly used procedures in that:

736

Eiometerja~s f991,

Vol 12 October

Once the cells have been identified, up to 30 parameters such as cell size and distribution can be measured. Errors such as misidentification of cell types will not (2) occur, due to the high specificity of the mo~~lonal staining technique. (3) As cell measurements are fully automated, a large number of cells can be analysed without counting errors being introduced.

(1)

ACKNOWLEDGEMENTS This staining regime has been developed under contract to the Laboratory of the Government Chemist, UK, with the objective of facilitating the quantitative assessment of biocompatibility and biosafety. The authors wish to acknowledge Peter Johnson, Professor of Immunology at the University of Liverpool, and the financial support of the Science and Engineering Research Council of the United Kingdom for the research studentship for John A. Hunt.

REFERENCES Iskandar, S.S., Emancipator, S.N. and Pretlow, T.G., Enzyme histochemistry of monocytes/macrophages. J. Histochem. Cytochem. 1989.37, 25-29 Hsu, S-M., Raine, L. and Fanger, H., Use of avidin-blotin-peroxidase complex tn immuno~roxidase techniques, J. ~~stochern. Cyfochem. 1981.29.577-580 Jouvin-Marche, E., Cerrlna, J., Coeffier, E., Duroux, P. and Benveniste, J., The effect of the Ca2+ antagonist nifedlpine on the release of platelet activating factor, slow reacting substance and ~-giucuronidase from human neutrophils, Eur. J. Pharmacol. 1983, 89, 19-26 Oppenheim, J.J. and Gery, I., lnterleukin 1 is more than an interleukin, immunology Today 1982, 3, 113-l 19 Dawson. W., Boot. JR, Walker. JR and Meade. C.J..Thearachldonic metabolltes. In Textbook of lmmunopharmocology (Eds M.M. Dale and J.C. Foreman). Black Scientific Publications,Oxford, UK, pp 126- 139 Dinarelio, C.A., interleukin 1, Rev. fnfect. Dis. 1984.

6, 5 l-95