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Time-related deviations of fibronectin and type I, II and III collagen on the interface between a hydroxyapatite disc and the rim of a calvarial trephine defect in rabbits
Biomateriak C!? 1996 Printed
in Great
17 (1996) Elsevier
Britain.
ELSEVIER
1515-1520
Science
Limited
All rights
reserved
0142-9612/96/$15.00
Time-related deviations of fibronectin and type I, II and III collagen on the interface between a hydroxyapatite disc and the rim of a calvarial trephine defect in rabbits T.C. Lindholm, T.J. Gao and T.S. Lindholm Bone Transplantation Research Group, Department F/N-33707, Tampere, fin/and To pursue
the events
in rabbits were
(diameter
around
hydroxyapatite
quantitatively
interface
determined
was noted
in the connective
control
was the finding
12th wk in contrast components
an increase study
indicated
have taken matrix with
in type
components host tissues
Keywords: Received
of exceptionally determined
I collagen
ahead
and the untreated
control
Hydroxyapatite,
with the implant. connective
tissue,
0
The change
in the CTHA interface
was confined
1996 Elsevier fibronectin,
III collagen
of connective derivation
collagen,
in these
proliferation
of
was parallel
from
tissue
to
8 to 16 wk. This
in CTHA interface
of connective
to the interaction
Science
in the CTHA
at the 8th and the
regeneration
The time-related
in the BHA and CTHA interface in contact
defect.
interface
and connective
due to an active
or characteristics
morphogenesis.
III collagen
and type
defects
in the BHA
of the staining
was probably
in fibronectin
in composition
tissue
of implanted
HA
Limited.
interface
1 February 1995; accepted 20 October 1995
changes in both the materials and host bone adjacent to refer to the interface. A few studies immunohistochemical changes in interfacial tissues during interaction between HA and surrounding bone or fibrous tissue. connective Our interest in immunohistochemical variations of interfacial tissues was further motivated by a study by Ripamonti7. Three months after implanting coral-derived HA cylinders (Interpore 600) intramuscularly in adult male baboons, detectable new bone was initiated in the implant without the addition of any osteoinductive factors. This may imply that change in the characteristics of fibrous connective tissue around the HA or interaction between HA and connective tissues contributed to bone generation. Type I collagen from 80% to 90% of the total collagen in bone and type II collagen is the major collagen present in cartilage and fibronectin, normally undetectable in bone, cartilage and dentin’-lo. We undertook experimental study to show this immunohistochemically determinable changes in
Synthetic hydroxyapatite (HA), with a crystalline structure similar to the mineral phase of bone, is considered to be biocompatible and has been commonly used as an osteoconductive bone substitute in dental surgery and as a coating material in orthopaedic One of the aspects in which many material surgery14. and biomedical scientists are commonly interested is what occurs in the interface between host tissues and HA material implanted into the living body. As known, the osteoconduction of HA in vivo requires close contact with the host bone to create a HA-bone interface which is a prerequisite to obtain reliable new bone ingrowth inside the HA. When there is more interstitial space between the HA and the host bone in the implanted site, the interface usually consists mainly of fibrous connective tissue5Y6. Intensive studies on the interface between different dimensions of HA implants and different locations of bone tissue have been carried out, most of them focussing on morphological or ingredient Correspondence
Bone
@HA)
staining
to the CTHA interface
and type
and a decrease
of bone
I collagen
one of the characteristics
compositions
in calvarial
and type I, II and III collagen
and host bone-HA
of type
high fibronectin
in the CTHA interface.
that variances
place
(CTHA)
enhancement
However,
to the BHA interface
immunohistochemically vascular
defect.
9 mm), implanted
of fibronectin
tissue-HA
A marked
University of Tampere, PO Box 607,
(diameter
changes
at the 12th and the 16th wk in comparison
in the untreated
interface
(HA) discs
11 mm), immunohistochemical
at 8, 12 and 16 wk postimplantation. tissue
of Clinical Medicine,
to Dr T. S. Lindholm.
1515
Biomaterials
1996,
Vol.
17
No. 15
Time-related
1516
deviations
of fibronectin
and collagen:
T.C.
Lindholm et al.
fibronectin and type I, II, III collagen on the interface between an implanted HA disc and the rim of a calvarial defect in the rabbit and to ascertain the events related to bone morphogenesis evoked by HA in fibrous connective tissue.
software: Microscale TM/TC Image Analysis System (Digithurst, Royston, UK). The results were expressed as a percentage of the measured area. The implanted HA, which was lost in the decalcification process, was not taken into account when measuring the areas.
MATERIALS
RESULTS
AND
METHODS
Coral-derived porous hydroxyapatite (Interpore 200, Interpore International, Irvine, CA, USA) was used as implant material in this study. This HA was made using a natural coral precursor (genus Porites). The HA block had a pore size ranging from 180-200pm with fully interconnecting channels. From this block, discs 9 x 1.3mm were prepared with a trocar with corresponding inner diameter. Six adult New Zealand white rabbits with an average body weight of 3.2 & 0.6 kg were used for the study. The rabbits were anaesthetized with Hypnorm’” (Leo Pharmaceuticals, Sweden) administered intramuscularly under spontaneous ventilation. The head was shaved and antiseptically cleaned. A 3 cm sagittal midline incision was made over the scalp of the animal to expose the temporal bone on both sides of the media suture. After subperiosteal undermining, two calvarial defects 11 mm in diameter were created symmetrically on bilateral sides of the midline using a manual trephine. Care was taken not to violate the dura. The prepared HA disc was placed on top of the dura in one of two defects and was not secured in the defect by other means than closing the periosteum and scalp in layers. The contralateral defect was left empty as a control. The discs inserted in the calvarial defects were not press-fitted because of the discrepancy between diameter of discs and size of defects. After killing at 8,12 and 16 wk, the cranium including the defect was excised en bloc, roentgenographed (Faxitron 805 X-ray system, McMinneville, OR, USA) and after dissection seen frontally with a band saw to obtain both half defects in the same specimen. The blocks were fixed in 10% neutral formalin, decalcified, embedded in paraffin and sectioned in 7 Alrn slices. The sections were stained with haematoxylin-eosin azure II for routine light microscopic observations and with immunohistochemical methods for analysis of type I, II and III collagens and fibronectin. Quantitative histometric analysis was used to determine the percentage of HA implant, new bone and connective tissue ingrowth at different times. The paraffin sections were immunohistochemically stained for type I, II and III collagen and fibronectin, the carried out by peroxidase-antiperoxidase methodl’. Antibodies to type I and III collagen were provoked in rabbit and goat, respectively, and activity confirmed by and specifity enzyme-linked immunosorbent assay (ELISA) before application”. Antibodies to type II collagen and fibronectin were obtained commercially (Dakopatts A/S, Glostrup, Denmark). immunohistochemically The stained sections were analysed by a computerized histomorphometry camera: JVC TK-870E CCD (Victor Co. Ltd, Japan), MicroEye TC grabbing and image processing card (Digithurst, Royston, UK) and Biomaterials 1996, Vol. 17 No. 15
Twelve trephine defects were harvested at three different observation times. The fusion of the rim of the defect and the HA disc was radiographically revealed in the site where bone intimately contacted the HA disc and radiolucent areas showed where fibrous connective tissue filled the space between the rim of the defect and the HA disc at 8 wk after implantation (Figure ~a and b). Newly formed bone and connective tissue ingrowth into the pores of the HA disc were microscopically observed. At 16 wk after implantation the entire HA disc was completely united with the host bone and the untreated defect showed bone regeneration starting from the circumference to about 50% of the defect area, the remaining part of the defect being covered by fibrous connective tissue (Figure 1~). The ratio between the HA disc and ingrowth of new bone and connective tissue in the defect was histomorphometrically quantified (Figure 2). Histologically, two patterns of interface were consistently recognized: (1) bone-HA (BHA) interface, where the HA disc bordered directly with the host bone (Figure 3), and (2) connective tissue-HA (CTHA) interface where the HA disc and the host bone were not in contact with each other (Figure 4). Newly formed bone in the BHA interface was mostly composed of cancellous or trabecular bone without cartilage. The connective tissue in the CTHA interface was characterized by an enriched vascular component with a cellular, loose connective tissue matrix, and condensation of connective tissue fibres bordered the surface of HA. Some osteocyte-like cells were seen in the tissue which had intermediate features between fibrous connective tissue and bone. The immunohistochemical staining to fibronectin and type III collagen in the BHA interface was weakly positive (1.68% and 6.50%, respectively) at the 8th wk. An increasing trend of fibronectin staining (4.35%) and a decreasing trend of type III collagen staining (5.45%) were observed at the 12th wk. The lowest positive staining for both was revealed at the 16th wk (2.38% and 2.85%, respectively). A marked increase in type I collagen staining was observed from the 12th wk (14.1%) to the 16th wk (34.87%) (Figure .!?a). The immunohistochemical staining for fibronectin and type III collagen in the CTHA interface was strongly positive (17.03% and 30.10%, respectively) at the 8th wk. The staining for fibronectin increased slightly (17.14%) by the 12th wk then decreased sharply (0.82%) by the 16th wk. The type III collagen staining decreased gradually from the 12th (16.9%) to the 16th wk (5.43%). The type I collagen staining was enhanced from the 12th wk (2.605%) to the 16th wk (11.42%) (Figure 5b). Compared to the immunohistochemical staining in the BHA interface, similar variations of fibronectin and
Time-related
deviations
of fibronectin
and collagen:
T.C. Lindholm
et a/.
1517
However, no detectable positive staining of the type II collagen was observed in the BHA, CTHA interface and connective tissue of the empty control at different times.
DISCUSSION
a
C
Figure 1 a, Radiographical image of a rabbit skull with two trephine defects on both sides of the median suture after 8 wk follow-up time. The HA block (HAb) shows good union on the lateral side of the defect while the medial side shows a radiolucent area between the defect margin and the block. The untreated control defect (Empty) on the right side shows partial healing around the rim of the defect. b, The CTHA interface shows strong staining of collagen type Ill (arrows) at 8 wk follow-up. HAb, Hydroxyapatite block; MS, median suture of the skull; E, Empty. Original magnification x5. c, The untreated control defect (E) shows only partial bone regeneration; fibrous connective tissue is seen in the central part of the defect. The block implanted defect shows bony contact (arrows) after 16wk follow-up. Original magnification x5.
type III collagen staining in connective tissue in the untreated defect were observed at the 8th wk (4.48% and 7.81%, respectively). The type III collagen staining rose by the 12th wk (11.34%) then slowed down by the 16th wk (3.69%). The lowest recognizable staining of fibronectin was seen at the 16th wk (0.09%). The type I collagen staining stepped up moderately from the 8th wk (5.85%) to the 16th wk (14.16%) (Figure 5~).
The calvarial defect represents an eminently suitable site for investigating regeneration of membranous bone as well as osteoconductive biomaterials. The spontaneous healing of a calvarial defect always proceeds from the edges towards the centre until complete bone bridging is achieved13. When complete bone bridging does not occur, but the defect heals by a fibrous union only, the defect may be termed a criticalsize-defect (CSD)13. When HA implants were used, the spontaneous healing process was shortened by the osteoconductivity of the implants. When the size of the HA block was well matched with the defect, rapid bone integration occurred”. If the size of the HA block did not match the defect, an extensive proliferated connective tissue filled in the gap between the HA block and the rim of the defect. The idea of using unmatched HA discs as implants in our study was that both bone-HA and connective tissue-HA interfaces were created and compared simultaneously in one defect so that it was possible to follow up what was going on in the connective tissue-HA interface when bone integration occurred in the bone-HA interface. After a HA disc was secured in the bone-HA interface at 8 wk, exaggerated proliferation of connective tissue invoked partially by migration of the HA disc was excluded. A time-related spontaneous change in connective tissue between HA and the rim of defect was demonstrated. A persistent and definitely enhanced staining of the type I collagen through the 12th to 16th wk showed an active synthesis of type I collagen in the BHA for new bone interface, which was evidence formation’. Osteoblasts are one of the dominant cells that produce and secrete type I collagen14. When good contact was obtained between the HA implant and the rim of the host calvarial defect, osteoblasts emigrated directly from the host bone, endosteum and periosteum to the substratum of the HA disc and laid collagens and other matrix proteins on the surface of the HA implant. As augmentation of the histologically visible new bone in the BHA interface, increased type I collagen was also noticed immunohistochemically. There was an apparent discrepancy in the amount of type I collagen between BHA and CTHA interface at both the 12th and 16th wk. This may imply that the effective expression of osteoinductivity in HA is confined to an intimate contact between host bone and HA implant. No type II collagen was found in either BHA or CTHA interface, which showed that the repair of a skull defect associated with HA implantation was a membranous rather than an endochondrial process. In contrast to the BHA interface and the connective tissue in the untreated defect, the finding of large amounts of fibronectin and type III collagen was one of the characteristics of connective tissue on the CTHA interface at the 8th and 12th wk. Fibronectin is mainly Biomaterials 1996, Vol. 17 No. 15
Time-related
1518
2 Quantitation by histomorphometry at 8, 12 and 16 wk after implantation
of new
bone,
Figure 3 Microphotograph of the interface area between the host bone and the HA block after 16 wk observation. Bony union is seen in the area with little connective tissue present (arrows). The HA block is partly filled with new bone and additional connective tissue. H, Host bone; HA, hydroxyapatite block. Haematoxylin-eosin-azur II, original magnification x40.
Figure 4 Microphotograph of the connective tissue interface after 8 wk follow-up. No direct contact between the host bone and the HA block can be observed at this site. H, Host bone, HA, hydroxyapatite block; CT, connective tissue interface. Haematoxylin-eosin-azur II, original magnification x40.
present in basement membranes of vascular walls and in soluble form in plasma158’fi. Type III collagen is a component of many connective tissues, particularly blood vessels’. The marked changes in these Biomaterials
1996, Vol. 17 No. 15
of fibronectin
hydroxyapatite
and collagen:
T.C. Lindholm
et al.
16 weeks
12 weeks
8 weeks Figure defects
deviations
and connective
tissue
in percentage
in calvarial
immunohistochemically determined compositions were considered to be a result of active proliferation of the vascular component in the connective tissue of the CTHA interface. Concomitant with reduction in type III collagen in the 12th wk, the proliferation of fibrovascular components was attenuated. This phenomenon was also evidenced morphologically. Since fibronectin shows an affinity for denatured collagens and other inflammatory products’“3*7, the fibronectin staining was higher until the 12th wk. The significance of the vascular proliferation in the CTHA interface initiated by implantation of HA was that it supplied a pathway for gathering undifferentiated mesenchymal cells and enriched oxygen in the local connective tissue, both of which are critical factors for membranous ossification. A biological role of fibronectin in the initial events of fibrovascular invasion has been assumed7,1”. A sharp enhancement of type I collagen, the main constructive collagen in bone, from the 12th to 16th wk, indicated bone regeneration in the CTHA interface. It might mark the beginning of transformation of the connective tissue in the CTHA interface to bone differentiation. Osteogenesis in porous hydroxyapatite implanted extraskeletallv was interpreted to be regulated by early morphogenesis condensation7. of the collagenous However, there might be a great variance in composition or characteristics of connective tissues in the CTHA interface before or at the same time as a morphological change could be observed. This studv yielded preliminary but clear evidence on derivation of immunohistochemistry in connective tissue around the HA implant. The molecular mechanisms that invoke the different changes in immunohistochemical staining in these interfaces are not known. It is probable that an early migration of the implanted HA disc facilitated more fibronectin and type III collagen formation in the CTHA interface. It is also possible for the porous HA to entrap different circulating or locally produced growth factors and cytokines that play different roles in orienting tissue-component differentiation’“. Whatever the mechanisms, the fact is that the time-related changes in components of the connective tissue matrix in the CTHA and BHA interface were eventually confined to the interaction between implanted HA and the different kinds of host tissue contacted by the implant. This may be assumed to be the basis of bone morphogenesis in connective tissue bordering on a HA implant.
Time-related
deviations
(4
of fibronectin
and collagen:
T.C. Lindholm
et a/.
Bone - HA interface
(b)
1519 Connective tissue - HA interface
50 7 45 -40 -35 -m 30 -$ “o 25 -*
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Time (weeks)
Time (weeks)
cc>
Control (untreated)
50 45
T
40 35
I
m 30 J “o 25 @ 20 I
8
16
12
Time (weeks) Figure 5 a, Results from the immunohistochemical staining of type I, II and Ill collagens and fibronectin in the bone-HA interface (BHA). Type I collagen shows a sharp increase in staining between the 12th and the 16th wk. n , Fibronectin; A, type I collagen; A, type II collagen; 0, type III collagen. b, Results from the immunohistochemical staining in the connective tissue-HA interface (CTHA). Type Ill collagen and fibronectin show a sharp decrease throughout the follow-up time. n , Fibronectin; A, type I collagen; a, type II collagen; 0, type III collagen. Results from the immunohistochemical staining in the untreated control defect. Type I collagen shows a steady increase in amount from 8 to 16 wk observations. n, Fibronectin; A, type I collagen; 0, type II collagen; 0, type Ill collagen.
ACKNOWLEDGEMENTS The authors wish to thank MS Ritva Sohlman, The Research Laboratory of the Invalid Foundation, Helsinki for the staining of histological samples, Matti Lehto MD and MS Toini Vieno, of the Paavo Nurmi Sports Medical Center, Turku, for organizing and carrying out the immunohistological staining, and Nikita Beliaev MSc (Eng) for aid with the computerized histomorphometry at the Institute of Dentistry, University of Turku, Finland.
Jarcho M. Calcium phosphate prosthetics. Clin Orthop 1981;
Kent JN, Quinn JH, Zide MF, Guerra LR, Boyne PJ. Alveolar ridge augmentation using nonresorbable hydroxyapatite with or without autogeneous cancellous bone. JOral Maxillofac Surg 1983; 41: 629-642.
3
Rosen HM, McFarland MM. The biologic behavior of hydroxyapatite implanted into the maxillofacial skeleton. Plast Reconstr surg 1990; 85: 718-723. Hofmann AA, Bachus KN, Bloebaum RD. Comparative study of human cancellous bone remodeling to titanium and HA-coated implant. / Arthroplasfy 1993; 8: 157-166. Holmes RE. Bone regeneration within a coralline hydroxyapatite implant. Plast Reconstr Surg 1979; 63: 626-633. Holmes RE, Hagler HK. Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: a histometric study. Plast Reconstr Surg 1988; 81: 662-671. Ripamonti U. The morphogenesis of bone in replicas of porous hydroxyapatite obtained from conversion of
4
5
6
REFERENCES 1
2
ceramics as hard 157: 259-278.
tissue
7
Biomaterials
1996, Vol. 17 No. 15
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1520
8
9
10
11
12
13
calcium carbonate exoskeletons of coral. ] Bone joint Surg1991; 73:692-703. Burgeson RE, Nimni ME. Collagen types. Molecular structure and tissue distribution. Clin Orthop 1992; 282:250-272. d’Ardenne JA, Burns J, Sykes BC, Kirkpatrick P. Comparative distribution of fibronectin and type III collagen in normal tissues. I Pathol 1983; 141:55-69. Engvall E, Ruoslahti E, Miller EJ. Affinity of fibronectin to collagens of different genetic types and to fibrinogen. JExp Med 1978; 147:1584. Sternberg LA. The unlabelled antibody peroxidaseantiperoxidase (PAP) method. In: Sternberg LA, ed. Immunohistochemisfry. New York: Wiley, 1979: 279321. Lehto M, Duance VC, Restall D. Collagen and fibronectin in a healing skeletal muscle injury. An immuno-histochemical study of the effects of physical activity on the repair of injured gastrocnemius muscle in the rat. J Bone Joint surg 1985; 7:820-828. Schmitz JP, Hollinger JO. The critical size defect as an
Biomaterials 1996. Vol. 17 No. 15
deviations
14
15
16
17
18
19
of fibronectin
and collagen:
T.C. Lindholm
et al.
experimental model for craniomandibular nonunions. Clin Orthop 1986;205:299-308. Holden CM, Bernard GW. Ultrastructural in vitro characterization of a porous hydroxyapatite/bone cell interface. J Oral Implant01 1990; 18:86-95. Kleinman HK, Wilkes CM, Martin GR. Interaction of fibronectin with collagen fibrils. Biochemistry 1981; 20:23-25. Mosher DF. Fibronectin. In: Spaet TH, ed. Progress in Hemostasis and Thrombosis. New York: Grune & Stratton, 1980: 111. Kurjinen M, Vaheri A, Robertson PJ, Stenman S. Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab Invest 1980; 43: 47-51. Weiss RE, Reddi AH. Role of fibronectin in collagenous matrix-induced mesenchymal cell proliferation and differentiation in viva. Exp Cell Bes 1981; 133:247-254. Wozney JM, Rosen V, Celeste AJ et al. Novel regulators of bone formation: molecular clones and activities. Science 1988;242:1528-1534.
in Great
17 (1996) Elsevier
Britain.
ELSEVIER
1515-1520
Science
Limited
All rights
reserved
0142-9612/96/$15.00
Time-related deviations of fibronectin and type I, II and III collagen on the interface between a hydroxyapatite disc and the rim of a calvarial trephine defect in rabbits T.C. Lindholm, T.J. Gao and T.S. Lindholm Bone Transplantation Research Group, Department F/N-33707, Tampere, fin/and To pursue
the events
in rabbits were
(diameter
around
hydroxyapatite
quantitatively
interface
determined
was noted
in the connective
control
was the finding
12th wk in contrast components
an increase study
indicated
have taken matrix with
in type
components host tissues
Keywords: Received
of exceptionally determined
I collagen
ahead
and the untreated
control
Hydroxyapatite,
with the implant. connective
tissue,
0
The change
in the CTHA interface
was confined
1996 Elsevier fibronectin,
III collagen
of connective derivation
collagen,
in these
proliferation
of
was parallel
from
tissue
to
8 to 16 wk. This
in CTHA interface
of connective
to the interaction
Science
in the CTHA
at the 8th and the
regeneration
The time-related
in the BHA and CTHA interface in contact
defect.
interface
and connective
due to an active
or characteristics
morphogenesis.
III collagen
and type
defects
in the BHA
of the staining
was probably
in fibronectin
in composition
tissue
of implanted
HA
Limited.
interface
1 February 1995; accepted 20 October 1995
changes in both the materials and host bone adjacent to refer to the interface. A few studies immunohistochemical changes in interfacial tissues during interaction between HA and surrounding bone or fibrous tissue. connective Our interest in immunohistochemical variations of interfacial tissues was further motivated by a study by Ripamonti7. Three months after implanting coral-derived HA cylinders (Interpore 600) intramuscularly in adult male baboons, detectable new bone was initiated in the implant without the addition of any osteoinductive factors. This may imply that change in the characteristics of fibrous connective tissue around the HA or interaction between HA and connective tissues contributed to bone generation. Type I collagen from 80% to 90% of the total collagen in bone and type II collagen is the major collagen present in cartilage and fibronectin, normally undetectable in bone, cartilage and dentin’-lo. We undertook experimental study to show this immunohistochemically determinable changes in
Synthetic hydroxyapatite (HA), with a crystalline structure similar to the mineral phase of bone, is considered to be biocompatible and has been commonly used as an osteoconductive bone substitute in dental surgery and as a coating material in orthopaedic One of the aspects in which many material surgery14. and biomedical scientists are commonly interested is what occurs in the interface between host tissues and HA material implanted into the living body. As known, the osteoconduction of HA in vivo requires close contact with the host bone to create a HA-bone interface which is a prerequisite to obtain reliable new bone ingrowth inside the HA. When there is more interstitial space between the HA and the host bone in the implanted site, the interface usually consists mainly of fibrous connective tissue5Y6. Intensive studies on the interface between different dimensions of HA implants and different locations of bone tissue have been carried out, most of them focussing on morphological or ingredient Correspondence
Bone
@HA)
staining
to the CTHA interface
and type
and a decrease
of bone
I collagen
one of the characteristics
compositions
in calvarial
and type I, II and III collagen
and host bone-HA
of type
high fibronectin
in the CTHA interface.
that variances
place
(CTHA)
enhancement
However,
to the BHA interface
immunohistochemically vascular
defect.
9 mm), implanted
of fibronectin
tissue-HA
A marked
University of Tampere, PO Box 607,
(diameter
changes
at the 12th and the 16th wk in comparison
in the untreated
interface
(HA) discs
11 mm), immunohistochemical
at 8, 12 and 16 wk postimplantation. tissue
of Clinical Medicine,
to Dr T. S. Lindholm.
1515
Biomaterials
1996,
Vol.
17
No. 15
Time-related
1516
deviations
of fibronectin
and collagen:
T.C.
Lindholm et al.
fibronectin and type I, II, III collagen on the interface between an implanted HA disc and the rim of a calvarial defect in the rabbit and to ascertain the events related to bone morphogenesis evoked by HA in fibrous connective tissue.
software: Microscale TM/TC Image Analysis System (Digithurst, Royston, UK). The results were expressed as a percentage of the measured area. The implanted HA, which was lost in the decalcification process, was not taken into account when measuring the areas.
MATERIALS
RESULTS
AND
METHODS
Coral-derived porous hydroxyapatite (Interpore 200, Interpore International, Irvine, CA, USA) was used as implant material in this study. This HA was made using a natural coral precursor (genus Porites). The HA block had a pore size ranging from 180-200pm with fully interconnecting channels. From this block, discs 9 x 1.3mm were prepared with a trocar with corresponding inner diameter. Six adult New Zealand white rabbits with an average body weight of 3.2 & 0.6 kg were used for the study. The rabbits were anaesthetized with Hypnorm’” (Leo Pharmaceuticals, Sweden) administered intramuscularly under spontaneous ventilation. The head was shaved and antiseptically cleaned. A 3 cm sagittal midline incision was made over the scalp of the animal to expose the temporal bone on both sides of the media suture. After subperiosteal undermining, two calvarial defects 11 mm in diameter were created symmetrically on bilateral sides of the midline using a manual trephine. Care was taken not to violate the dura. The prepared HA disc was placed on top of the dura in one of two defects and was not secured in the defect by other means than closing the periosteum and scalp in layers. The contralateral defect was left empty as a control. The discs inserted in the calvarial defects were not press-fitted because of the discrepancy between diameter of discs and size of defects. After killing at 8,12 and 16 wk, the cranium including the defect was excised en bloc, roentgenographed (Faxitron 805 X-ray system, McMinneville, OR, USA) and after dissection seen frontally with a band saw to obtain both half defects in the same specimen. The blocks were fixed in 10% neutral formalin, decalcified, embedded in paraffin and sectioned in 7 Alrn slices. The sections were stained with haematoxylin-eosin azure II for routine light microscopic observations and with immunohistochemical methods for analysis of type I, II and III collagens and fibronectin. Quantitative histometric analysis was used to determine the percentage of HA implant, new bone and connective tissue ingrowth at different times. The paraffin sections were immunohistochemically stained for type I, II and III collagen and fibronectin, the carried out by peroxidase-antiperoxidase methodl’. Antibodies to type I and III collagen were provoked in rabbit and goat, respectively, and activity confirmed by and specifity enzyme-linked immunosorbent assay (ELISA) before application”. Antibodies to type II collagen and fibronectin were obtained commercially (Dakopatts A/S, Glostrup, Denmark). immunohistochemically The stained sections were analysed by a computerized histomorphometry camera: JVC TK-870E CCD (Victor Co. Ltd, Japan), MicroEye TC grabbing and image processing card (Digithurst, Royston, UK) and Biomaterials 1996, Vol. 17 No. 15
Twelve trephine defects were harvested at three different observation times. The fusion of the rim of the defect and the HA disc was radiographically revealed in the site where bone intimately contacted the HA disc and radiolucent areas showed where fibrous connective tissue filled the space between the rim of the defect and the HA disc at 8 wk after implantation (Figure ~a and b). Newly formed bone and connective tissue ingrowth into the pores of the HA disc were microscopically observed. At 16 wk after implantation the entire HA disc was completely united with the host bone and the untreated defect showed bone regeneration starting from the circumference to about 50% of the defect area, the remaining part of the defect being covered by fibrous connective tissue (Figure 1~). The ratio between the HA disc and ingrowth of new bone and connective tissue in the defect was histomorphometrically quantified (Figure 2). Histologically, two patterns of interface were consistently recognized: (1) bone-HA (BHA) interface, where the HA disc bordered directly with the host bone (Figure 3), and (2) connective tissue-HA (CTHA) interface where the HA disc and the host bone were not in contact with each other (Figure 4). Newly formed bone in the BHA interface was mostly composed of cancellous or trabecular bone without cartilage. The connective tissue in the CTHA interface was characterized by an enriched vascular component with a cellular, loose connective tissue matrix, and condensation of connective tissue fibres bordered the surface of HA. Some osteocyte-like cells were seen in the tissue which had intermediate features between fibrous connective tissue and bone. The immunohistochemical staining to fibronectin and type III collagen in the BHA interface was weakly positive (1.68% and 6.50%, respectively) at the 8th wk. An increasing trend of fibronectin staining (4.35%) and a decreasing trend of type III collagen staining (5.45%) were observed at the 12th wk. The lowest positive staining for both was revealed at the 16th wk (2.38% and 2.85%, respectively). A marked increase in type I collagen staining was observed from the 12th wk (14.1%) to the 16th wk (34.87%) (Figure .!?a). The immunohistochemical staining for fibronectin and type III collagen in the CTHA interface was strongly positive (17.03% and 30.10%, respectively) at the 8th wk. The staining for fibronectin increased slightly (17.14%) by the 12th wk then decreased sharply (0.82%) by the 16th wk. The type III collagen staining decreased gradually from the 12th (16.9%) to the 16th wk (5.43%). The type I collagen staining was enhanced from the 12th wk (2.605%) to the 16th wk (11.42%) (Figure 5b). Compared to the immunohistochemical staining in the BHA interface, similar variations of fibronectin and
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However, no detectable positive staining of the type II collagen was observed in the BHA, CTHA interface and connective tissue of the empty control at different times.
DISCUSSION
a
C
Figure 1 a, Radiographical image of a rabbit skull with two trephine defects on both sides of the median suture after 8 wk follow-up time. The HA block (HAb) shows good union on the lateral side of the defect while the medial side shows a radiolucent area between the defect margin and the block. The untreated control defect (Empty) on the right side shows partial healing around the rim of the defect. b, The CTHA interface shows strong staining of collagen type Ill (arrows) at 8 wk follow-up. HAb, Hydroxyapatite block; MS, median suture of the skull; E, Empty. Original magnification x5. c, The untreated control defect (E) shows only partial bone regeneration; fibrous connective tissue is seen in the central part of the defect. The block implanted defect shows bony contact (arrows) after 16wk follow-up. Original magnification x5.
type III collagen staining in connective tissue in the untreated defect were observed at the 8th wk (4.48% and 7.81%, respectively). The type III collagen staining rose by the 12th wk (11.34%) then slowed down by the 16th wk (3.69%). The lowest recognizable staining of fibronectin was seen at the 16th wk (0.09%). The type I collagen staining stepped up moderately from the 8th wk (5.85%) to the 16th wk (14.16%) (Figure 5~).
The calvarial defect represents an eminently suitable site for investigating regeneration of membranous bone as well as osteoconductive biomaterials. The spontaneous healing of a calvarial defect always proceeds from the edges towards the centre until complete bone bridging is achieved13. When complete bone bridging does not occur, but the defect heals by a fibrous union only, the defect may be termed a criticalsize-defect (CSD)13. When HA implants were used, the spontaneous healing process was shortened by the osteoconductivity of the implants. When the size of the HA block was well matched with the defect, rapid bone integration occurred”. If the size of the HA block did not match the defect, an extensive proliferated connective tissue filled in the gap between the HA block and the rim of the defect. The idea of using unmatched HA discs as implants in our study was that both bone-HA and connective tissue-HA interfaces were created and compared simultaneously in one defect so that it was possible to follow up what was going on in the connective tissue-HA interface when bone integration occurred in the bone-HA interface. After a HA disc was secured in the bone-HA interface at 8 wk, exaggerated proliferation of connective tissue invoked partially by migration of the HA disc was excluded. A time-related spontaneous change in connective tissue between HA and the rim of defect was demonstrated. A persistent and definitely enhanced staining of the type I collagen through the 12th to 16th wk showed an active synthesis of type I collagen in the BHA for new bone interface, which was evidence formation’. Osteoblasts are one of the dominant cells that produce and secrete type I collagen14. When good contact was obtained between the HA implant and the rim of the host calvarial defect, osteoblasts emigrated directly from the host bone, endosteum and periosteum to the substratum of the HA disc and laid collagens and other matrix proteins on the surface of the HA implant. As augmentation of the histologically visible new bone in the BHA interface, increased type I collagen was also noticed immunohistochemically. There was an apparent discrepancy in the amount of type I collagen between BHA and CTHA interface at both the 12th and 16th wk. This may imply that the effective expression of osteoinductivity in HA is confined to an intimate contact between host bone and HA implant. No type II collagen was found in either BHA or CTHA interface, which showed that the repair of a skull defect associated with HA implantation was a membranous rather than an endochondrial process. In contrast to the BHA interface and the connective tissue in the untreated defect, the finding of large amounts of fibronectin and type III collagen was one of the characteristics of connective tissue on the CTHA interface at the 8th and 12th wk. Fibronectin is mainly Biomaterials 1996, Vol. 17 No. 15
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2 Quantitation by histomorphometry at 8, 12 and 16 wk after implantation
of new
bone,
Figure 3 Microphotograph of the interface area between the host bone and the HA block after 16 wk observation. Bony union is seen in the area with little connective tissue present (arrows). The HA block is partly filled with new bone and additional connective tissue. H, Host bone; HA, hydroxyapatite block. Haematoxylin-eosin-azur II, original magnification x40.
Figure 4 Microphotograph of the connective tissue interface after 8 wk follow-up. No direct contact between the host bone and the HA block can be observed at this site. H, Host bone, HA, hydroxyapatite block; CT, connective tissue interface. Haematoxylin-eosin-azur II, original magnification x40.
present in basement membranes of vascular walls and in soluble form in plasma158’fi. Type III collagen is a component of many connective tissues, particularly blood vessels’. The marked changes in these Biomaterials
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16 weeks
12 weeks
8 weeks Figure defects
deviations
and connective
tissue
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in calvarial
immunohistochemically determined compositions were considered to be a result of active proliferation of the vascular component in the connective tissue of the CTHA interface. Concomitant with reduction in type III collagen in the 12th wk, the proliferation of fibrovascular components was attenuated. This phenomenon was also evidenced morphologically. Since fibronectin shows an affinity for denatured collagens and other inflammatory products’“3*7, the fibronectin staining was higher until the 12th wk. The significance of the vascular proliferation in the CTHA interface initiated by implantation of HA was that it supplied a pathway for gathering undifferentiated mesenchymal cells and enriched oxygen in the local connective tissue, both of which are critical factors for membranous ossification. A biological role of fibronectin in the initial events of fibrovascular invasion has been assumed7,1”. A sharp enhancement of type I collagen, the main constructive collagen in bone, from the 12th to 16th wk, indicated bone regeneration in the CTHA interface. It might mark the beginning of transformation of the connective tissue in the CTHA interface to bone differentiation. Osteogenesis in porous hydroxyapatite implanted extraskeletallv was interpreted to be regulated by early morphogenesis condensation7. of the collagenous However, there might be a great variance in composition or characteristics of connective tissues in the CTHA interface before or at the same time as a morphological change could be observed. This studv yielded preliminary but clear evidence on derivation of immunohistochemistry in connective tissue around the HA implant. The molecular mechanisms that invoke the different changes in immunohistochemical staining in these interfaces are not known. It is probable that an early migration of the implanted HA disc facilitated more fibronectin and type III collagen formation in the CTHA interface. It is also possible for the porous HA to entrap different circulating or locally produced growth factors and cytokines that play different roles in orienting tissue-component differentiation’“. Whatever the mechanisms, the fact is that the time-related changes in components of the connective tissue matrix in the CTHA and BHA interface were eventually confined to the interaction between implanted HA and the different kinds of host tissue contacted by the implant. This may be assumed to be the basis of bone morphogenesis in connective tissue bordering on a HA implant.
Time-related
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(4
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et a/.
Bone - HA interface
(b)
1519 Connective tissue - HA interface
50 7 45 -40 -35 -m 30 -$ “o 25 -*
20 -,I
15-.
15 --
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12
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Time (weeks)
Time (weeks)
cc>
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8
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12
Time (weeks) Figure 5 a, Results from the immunohistochemical staining of type I, II and Ill collagens and fibronectin in the bone-HA interface (BHA). Type I collagen shows a sharp increase in staining between the 12th and the 16th wk. n , Fibronectin; A, type I collagen; A, type II collagen; 0, type III collagen. b, Results from the immunohistochemical staining in the connective tissue-HA interface (CTHA). Type Ill collagen and fibronectin show a sharp decrease throughout the follow-up time. n , Fibronectin; A, type I collagen; a, type II collagen; 0, type III collagen. Results from the immunohistochemical staining in the untreated control defect. Type I collagen shows a steady increase in amount from 8 to 16 wk observations. n, Fibronectin; A, type I collagen; 0, type II collagen; 0, type Ill collagen.
ACKNOWLEDGEMENTS The authors wish to thank MS Ritva Sohlman, The Research Laboratory of the Invalid Foundation, Helsinki for the staining of histological samples, Matti Lehto MD and MS Toini Vieno, of the Paavo Nurmi Sports Medical Center, Turku, for organizing and carrying out the immunohistological staining, and Nikita Beliaev MSc (Eng) for aid with the computerized histomorphometry at the Institute of Dentistry, University of Turku, Finland.
Jarcho M. Calcium phosphate prosthetics. Clin Orthop 1981;
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