Degradationof and tissue reaction to

biodegradablepoly(Llactide) for use

as internal fixation of fractures:
a study in rats
R.R.M.Bos*,F.R. Rozema”,G. Boering”,A.J. Nijenhuis?,
A.J. Pennings?,A.B.Verwey?,P. Nieuwenhuisfand H.W.B.Jansent
Departments of Oral and Maxillofacial Surgery*, Polymer Chemistry? and Histology and Cell 8iologyz, University of Groningen,
The Netherlands
(Received 10 August 1989; revised 16 September 1989; accepted 15 November 1989)

Samples of high-molecular-weight poly(t-lactide) (PM) (ic?, = 9.0 X 1 05), a biomaterial developed for
plates and screws used in internal fixation of jaw fractures, were implanted subcutaneously in the backs of
rats to study tissue reaction to PLLA and to follow the degradation process. The P1l.A seemed to follow the
degradation pattern typical of biodegradable polyesters. After pure hydrolysis up to about 104 wk.
phagocytic activity of macrophages was found at about 143 wk. Full resorption of PM was not
demonstrated in this study. Except for the early and final parts of the implant period, no acute or chronic
inflammatory reaction was observed. No implant was rejected. It is estimated that more than 3 yr will be
required for total resorption of PM. For bone-healing this long period is of no practical importance. There
is no need for removal of P1l.A after fracture healing as is the case with metal fixation devices. Thus, PLLA
has potential application in internal fixation of fractures and osteotomies in the maxillofacial region and
other fractures that are not too heavily loaded in the human body.

Keywords: Implants, biodegradation, fracture fixation

Internal fixation of osseous fractures with metallic plates and
screws is generally accepted and reliable. However, these
metallic implants must be removed after the fracture has
healed. A stress-protection effect occurs beneath the plates
causing osteopenia of the underlying bone and resulting in
spontaneous fractures under normal loading. In addition,
inflammatory reactions occur due to loosening and corrosion
of the metallic deviceslm7.
Metallic implants cause backscattering in patients
irradiated after bony reconstruction because of remnants of a
malignant tumour. These implants also cause artefacts in
computerized tomography and magnetic resonance
imaging’, ‘. Some metals (mercury, gold, platinum, beryllum,
chromium and nickel) cause allergic reactions that can vary
from eczema toanaphylactic shock”. Cobalt, chromium and
nickel are haptens that are generally used in the alloys from
which plates and screws for fixation purposes are manu-
factured. Titanium and molybdenum are not believed to be
haptens. The immune role of vanadium remains unclear.
Chromium, nickel and cobalt are potent carcinogens in
Correspondence to Dr R.R.M. Bos, Department of Oral and Maxillofacial
Surgery, University of Groningen, PO Box 72, 9700 RB Groningen, The
Netherlands.
animals. Titanium is not known to be carcinogenic in
animals, whereas the evidence concerning vanadium is
inconclusive. Only a few studies on implant site tumours in
human patients associated with stainless steel fracture
fixation devices are reported”.
For these reasons metallic plates and screws, used for
internal fixation of jaw fractures or osteotomies in our work
are routinely removed. This means a second operation with
all its costs and extra discomfort for the patient.
Plates and screws manufactured from biocompatible,
biodegradable material with appropriate load-bearing
properties and degradation rate could obviate the necessity
and costs of a second operation for their removal.
In previous papers we reported the successful appli-
cation of plates and screws of poly(L-lactide) for internal
fixation of artificially created mandibular fractures in sheep
and dogs”-‘4. As poly(L-lactide) is considered to be a fully
resorbable biocompatible materia17,‘5-26, we used it in
specially designed plates and screws for the internal fixation
of unstable zygomatic fractures in 10 patientsz7.
This study monitored the degradation of poly(~-
lactide) over time and the reaction of the surrounding tissues
during degradation.

0 199 1 Butterworth-Heinemann Ltd. 0142-96 12/9 1/010032-05

32 Biomaterials 1991, Vol 12 January


MATERIAL AW METHODS
Polyft-lactide) was synthesized at a low catalyst concentration
of 0.015 wt% stannous-2-ethylhexanoate (Sigma Chem.
Corp., USA) and 100°C as described previously”. The final
material was microporous, with pore sizes in the range
50- 100 nm. Molecular weight M, was 9.0 X 1 05.
Plates of 8 X 8 X 2 mm were machined out of a block
of PLLA. All edges were rounded off. A hole of 1.5 mm
diameter was made in the centre of the plate to enable tissue
ingrowth. The plates were sterilized with ethylene oxide and
evacuated under high vacuum (10e5 Torr) for 24 h. They
were then implanted subcutaneously in the backs of 35
female Wistar Albino rats (TNO, Zeist, The Netherlands),
about 6 wk old and 2009 in weight. Under general
anaesthesia (nitrous oxide-oxygen-fluothane) the backs of
the rats were shaved and disinfected with iodine solution.
Under sterile conditions a skin incision was made in the
middle of the back and a subcutaneous pocket was created
by blunt preparation with a pair of scissors. One plate was
inserted into this pocket (Figure la), after which the skin
was closed using two to three Dexon ‘S’ 3-O sutures
(Cyanamid, Gosport, Hampshire, UK). Aftertheoperation the
rats had free access to standard rat food and water.
Clinical follow-up was planned up to 104 wk post-
operatively. The longest period of observation eventually
was 143 wk. At regular time intervals two rats were killed.
After determining the localization, plate and surrounding
soft tissue were excised en bloc with scalpel and scissors
(figure 16). Subsequently the PLLA plate was carefully
removed from the soft tissue covering after incising this
tissue over one edge of the plate.
Figure 1 Implantation of a PLLA plate in a subcutaneous pocket at the
back of a rat (a) and removal of this plate and surrounding soft tissue 1 wk
after implantatron (b).
Poly(i-IactideJ in mtemal fracture f/xation. R.R.M. 80s et a/
Histological sections were made of the surrounding
soft tissues for light microscopic analysis after fixation in 4%
formaldehyde and paraffin embedding. Sections were
stained with haematoxylin and eosin according to standard
procedures.
The PLLA plates removed were carefully rinsed with
saline solution and then stored in ethanol 70% for 24-48 h.
The plates were then dried under vacuum (20 Torr) over
Siccapent@ (5. Merck, Darmstadt, FRG) to constant weight.
An Ubbelohde viscometer (type Oaf was used to determine
intrinsic viscosity [r)] of the polymer in chloroform at 25°C.
The viscosity average molecular weight (A/Id of the samples
was estimated from: [r)] = 5.45 X 1 0e4. IIJ~‘.‘~.
RESULTS
Macroscopic findings
All animals recovered well from the implantation operation.
Clinically there was no sign of inflammatory reaction nor
plate rejection. Integral removal of the plates from the
surrounding soft tissue was possible up to 78 wk. They
were easily removed from a smooth capsule. No difference in
form was detected. After 91 wk and later the plates had
become so brittle that it was not possible to remove them in
one piece. Fragments were collected only for determination
of the average molecular weight M,. Determination of mass
loss was not possible because little particles of PLLA were
left behind in the capsule so as not to damage surrounding
soft tissue needed for histological examrnation.
Decrease in molecular weight and mass loss
Figure 2 shows the decrease in molecular weight and the
mass loss in time; data are number averages of values for
each pair of rats killed at each time interval. Individual values
for each pair were almost equal. The decrease in molecular
weight in the first 3 month was very rapid and then passed off
more gradually. Mass loss was only observed from 26 wk on.
Histological findings
In about 12 wk a connective tissue capsule had developed
around the PLLA material. At week 1 (Figure 3a) a
homogeneous tissue layer about 200 pm thick and mainly
consisting of thin collagen fibres with numerous fibroblasts
and fibrocytes in between was found around the PLLA
implants. In this layer a capillary network is present and a
40 60
Tome I weeks)
Figure 2 Decrease in M, (e) and mass loss (0) of subcutaneous
implanted PLEA (M, = 9 x 105j.
Biomaterials 139 f. Vol 12 January 33
PolyfL-la&de) in intern& fracture fixation: R.R.M. Bos et a!.

Figure 3 Phot~mic~ograpbs showing the interface between the PM& imptant {P) and the surround&g soft tissue. (Origirrd mag~if~~a~~o# X 200.) (8) Week f:
toose connective tissue facing the PLEA implant. (b) Week 2: dense layer of about 5 ceiis in thickness mainly consisting of macrophages surrounded by loose
connective tissue with an adjacent capillary network. (c) Week 12: dense connective tissue directly facing the PLLA implant. (d) Week 52: mature dense fibrous
capsule facing the PLLA implant. (e) Week 104: mature dense fibrous capsule in contact with the PLlA implant. Remnants of PLEA present.

very mild inflammatory reaction with some macrophages contact with the PLLA plate. Surrounding this layer was a
and only a few mononuclear cells and plasma cells were fairly homogeneous layer rich in capillaries, fibroblasts and
found. young loose collagen. In the next period, up to 12 wk, the
At week 2 (Figure 3b) there was a compact layer, compact layer disappeared. At week 12 (figure 3c) a
about 5 cells thick (predominantly macrophages) in direct homogeneous layer about 200,~m thick was seen around

34 Biomaterials T99 I, Vol 12 January

 Poly(i-lactide) m tntemaf fracture f/rat/on: R.R.M. Bos ef al.

the PLLA with evenly distributed fibrocytes and collagen considering the very low cell density at the interface of the
fibres running parallel to the PLLA surface. Capillaries were PLLAwith the surrounding capsule, pure hydrolysis causing
less striking. very little irritation must be responsible for the decrease in
In the following period up to 52 wk a further ripening molecular weight and for the mass loss at the beginning, at
occurred of the homogeneous fibrous capsule with only a least up to 104 wk after implantation. The dense cell layer
few spindle-shaped fibrocytes (Figure 3d). From week 52 mainly consisting of macrophages in direct contact with the
on the capsule around the PLLA became thinner again. At PLLA found in the first few weeks after implantation can be
week 104 (Figure 3e) a thin fibrous capsule of average ascribed to an inflammatory reaction due to the surgical
thickness 125pm was visible in close contact with the trauma of the implantation procedure combined with an
PLLA. initial foreign body tissue reaction30.
At week 104 only one of the 35 operated rats was left: The mass loss, as observed from 26 wk on, indicated
it had to be killed at 143 wk because of a mammary that resorption of PLLA had started. This, however, did not
fibroadenoma. The microscopic image of the capsule around give rise to any histologically detectable reaction up to
the PLLA had changed considerably (Figure 4a). The 104 wk, illustrating the biocompatibility of PLLA and its
collagenous capsule had thickened to about 200 pm. At the degradation products.
interface with the PLLA there was a layer of irregular Somewhere between 104 and 143 wk. cellular
thickness with foamy macrophages. Between these macro- activity returns as a final stage of the degradation of the PLLA
phages, particles of PLLA could be detected. Giant cells were material, wrth the presence of foamy macrophages at
not present. There was a noticeably higher cell density on the 143 wk. Apparently these cells are clearing away the
skin side of the PLLA implant than on the ventral side. The particles of PLLA which have been released from the
highest cell density was seen in the hole made in the centre implanted plate. Giant cells were not observed by 143 wk; it
of the implanted plates (Figure #b). Between these cells, cannot be excluded that these cells might be found at a later
implant-derived particles could be detected. stage of PLLA degradation. The higher ceil density on the
skin side and in the hole of the PLLA plate at 143 wk may be
due to mechanically induced release of particles of PLLA, the
DISCUSSION ventral side being more protected from direct forces like
scratching or rubbing. At present it IS not clear how much
Earlier studies showed rapid decrease in molecular weight of time it will take for total resorption of PLLA. Since giant cells
PLLA In the first 3 month after implantation”-‘4, and we were not yet present at 143 wk post-operatively, the final
initially estimated that PLLA implants would be resorbed in stage of degradation may not have been reachedz3 31. We
about 12-I 8 monthz7. Therefore an extensive follow-up estimate that it will take more than 3 yr for total resorption of
study (up to 104 wk) was undertaken using rats. The tissue PLLA.
intervals planned required at least 28 of the 35 rats to be With exception of the early part implant period there
operated upon; an excess of 7 rats was used to compensate was no microscopic sign of chronic or acute inflammatory
for loss of animals over 2 yr due to various illnesses and reactions, until the 143 wk rat. The reappearance of
tumour growth28~2g, Only one rat survived the 2 yr period: macrophages, that participated in the final part of the
28 were used for the various tests, 2 died for unknown process of degradation of the PLLA, represents a foreign
reasons and 4 were killed because of mammary tumours. body tissue reaction, a concomitant rather mild but chronic
mammary tumours are common in female rats, especially inflammato~ reaction that could not be detected clinically.
those over 24 month. Tumour incidence in this study was The observed degradation of and tissue reaction to
comparable with that reported in the literature28. The last rat PLLA seems comparable with that reported else-
where% 7 15-26.31, At initial stages, degradation of PLLA
was killed after 143 wk.
Comparison of histological findings with the decrease takes place predominantly in the amorphous regrons by
In molecular weight and the mass loss suggests that, random and autocatalytic hydrolyses. At a later stage, the

FIgwe 4 Pbotomicrogra~hs show;ng the interface between the FLL4 imp/ant (PJ and the surrou~~d/ng soft tissue at week 143. (a) Foamy macropbages tn
contact with the PLUI implant. (Orisinat magnifkatwn X 200 J (6) In the hole area Imp~aRt-deflved PLEA pan/c/es (pJ are present m between and surrounded by
foamy macrophages. (Original magnificarion X 320.)

B/omatenals lY91. Vol 12 January 35

Poly(L-lactide) in internal fracture fixation: R.R.M. Bos et al
remaining highly crystalline PLLA hydrolyses at a much
slower rate. At an even later stage, degradation has
progressed so far that small particles are shed from the main
body of the implant and subsequently removed by phago-
cytic activity of macrophages and probably later lytic activity
of giant cells.
The degradation pattern of PLLA suggests that it
would still be present long after bone healing has been
completed. PLLA, however, also lost its mechanical
properties for a considerable time”-‘4. For bone healing the
long degradation time of PLLA is not important since the
stress-protection effect will have disappeared. However, it
would be preferable if, as soon as bone healing is completed,
the PLLA material disappeared quickly. This would be
especially advantageous where the osteosynthesis is situated
such that the patient can palpate the plates and screws, for
example at the corner of the eye socket.
This study did not show complete degradation of
PLLA. However, the degradation pattern and the tissue
reaction observed seem parallel to that reported in the
literature. Further research will be aimed at acceleration of
the degradation of PLLA after 6 wk of fracture healing
without spoiling the favourable mechanical and implant
properties necessary to enable undisturbed bone healing.
ACKNOWLEDGEMENTS
The authors acknowledge the contributions of G. Boezerooij,
R. Dijkstra and D. Huizinga.
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