The implantation of cartilaginous and periosteal tissue into growth plate defects

International Orthopaedics (SICOT) (1994) 18: 220- 228 International Orthopaedics

© Springer-Verlag 1994

The implantation of cartilaginous and periosteal tissue into growth plate defects

T. Wirthl, S. Byersl, R. W. Byard 1, J. J. Hopwood 2, and B. K. Foster 1

Departments of Orthopaedic Surgery 1, Histopathology and Chemical Pathology 2, The Adelaide Childrens' Hospital, Australia

Accepted: 23 November 1993

Summary. This experimental study reports the results of implantation o f cartilaginous and periosteal tissues into growth plate defects in the tibiae of sheep. When no material was used, the defect rapidly filled with marrow-like tissue. When cartilage from the margin of the secondary centre of ossification was implanted, endochondral ossification continued and no shortening or deformity resulted. Implantation of periosteum with or without reconstructed peripheral tissues resulted in the formation of a bony bridge which led to a 32% inhibition o f longitudinal growth and a 12 ° varus deformity in the absence of peripheral connective tissues. After reconstruction with these tissues, the inhibition of longitudinal growth was 47% with a 28 ° varus deformity. The chondroprogenitor cells in the implanted tissues cannot change phenotypic expression. Periosteum has a strong potential for bone formation after it has been implanted.

R~sumC L'excision d'une plaque de croissance partiellement fusionn(e et son remplacement par interposition de diff(rents tissus n'a permis de montrer ni re-formation ni r(paration de la structure anatomique de cette zone. Dans cette (rude exp(rimentale nous pr(sentons les r(sultats de l'implantation de cartilage ou de p~rioste dans la perte de substance cr(~e au niveau de la partie interne de la plaque de croissance tibiale sur un modkle animal ovin. Sans interposition la cavit~ est rapidement remplie par un tissu ayant quelques

Reprint requests to: T. Wirth, Department of Orthopaedics, Phillips-University Marburg, Baldingerstrasse, D-35033 Mar- burg, Germany

ressemblances avec la mo~lle osseuse. Le cartilage, gtla limite du centre secondaire d'ossification, continue le processus d'ossification en- chondrale avec formation d'os nouveau; plus lentement cependant que dans la zone de croissance normale adjacente. Sans comblement, de m~me qu'aprOs implantation de cartilage, il ne se produit ni raccourcissement, ni angulation du membre op(r(. L'implantation de p(rioste, avec ou sans reconstruction des structures p(riph(riques entrafne la formation d' un pont osseux notable. II y a une inhibition de la croissance en longueur de 32% et une angulation en varus de 12 ° en l'absence de reconstruction des tissus p(riph- (riques. Il y a une inhibition de la croissance de 47% et une angulation de 28 °dans l'(ventualit( inverse. Nous en concluons que les cellules chon- droprog(niques du tissu implant( ne peuvent pas changer leur expression ph(notypique. Le p(rioste a un potentiel remarquable pour induire la formation d'os nouveau aprOs transposition.

Introduction

The surgical treatment of partial epiphyseal growth arrest due to trauma or infection by resection of the bony bridge and interposition of autologous fat was first demonstrated by Langenskj61d [20]. Other materials, such as silicone or methylmethacrylate, have also been successfully used for interposition [2, 3, 4, 18, 44]. The beneficial effect of these procedures was the prevention of the reformation of the bony bridge, but no convincing

T. Wirth et al.: Cartilaginous and periosteal implants in growth plate defects 221

Fig. 1 a - d . Photomicrographs (xl.8) of specimens taken immediately after operation, a After an empty defect; b after implantation of the hyaline cartilage of the secondary centre of ossification and adjacent zone of Ranvier; c after implantation of a free flap of periosteum; d after a free periosteal graft with peripheral connective tissues attached

reformation or repair of growth plate cartilage has ever been demonstrated [10, 21].

The implantation of cartilage and cultured chondrocytes has also been used in animal experiments to prevent recurrence of the bony bridges [1, 2, 8, 9, 10, 13, 16, 22, 24, 33, 45]. Cartilage implants were found to be better than fat grafts for preventing shortening of the limb and angular deformity because of an inhibitory effect on bone [24, 33]. Cultured chondrocytes survived and showed some organisation into columns, but produced a strong immunological response by the host tissue [11, 16].

The different regions which contribute to over- all growth must be distinguished in order to un- derstand the complex mechanism of bone growth. Firstly, longitudinal bone growth is produced by the cartilage of the growth plate with its func- tionally differentiated layers [5]. Secondly, growth in width is brought about by the peripherally lo- calised zone of Ranvier and the deep layer of endochondral ossification in the articular cartilage of the epiphysis. The zone of Ranvier consists of 3 layers [39], the innermost, which has densely packed cells which are the osteoblast progenitor cells, the intermediate, with loosely packed cells

222 T. Wirth et al.: Cartilaginous and periosteal implants in growth plate defects

which synthesise protoglycans and are probably chondroprogenitor cells [14, 39, 41], and an outer fibrous layer, which gives circumferential support to the growth plate [19, 35].

Periosteum has been described as differentiating into both cartilage and bone during fracture heal- ing, and during this process the periosteal mesenchymal progenitor cells are thought to differentiate into chondrocytes and osteoblasts [7, 37]. The osteochondrogenic potential of the periosteum became evident when periosteum was implanted into full thickness defects of articular cartilage, and was followed by the development of hyaline cartilage [29]. Cartilage and bone formation have also been seen after subcutaneous injection of cultured periosteal cells in mice [28].

The present study was set up using an ovine model [9] to find out whether cells with chondrogenic potential were capable of providing growth plate repair. In artificially created defects in growth plates of immature sheep periosteum, en- dochondrally ossifiying cartilage from the secondary centre of ossification and zone of Ranvier tissue were implanted and their capability of repair examined.

Materials and methods

Defects were created at the margin of the medial aspect of the proximal tibial growth plate of 20 immature six-week old Merino sheep, weighing 11.5 to 20 kg; both tibiae were used.

Anaesthesia was induced with pentothal (25 mg/kg) followed by halothane and nitrous oxide. An anteromedial approach was made to expose the proximal epiphyseal plate; the perichondrium was peeled off with the attached hyaline cartilage of the secondary centre of ossification and the adjacent zone of Ranvier. In both legs, a peripheral defect measuring 10 mm x 5 mm x 2 mm (width x depth x height) was made on the medial side of the tibia with a dental burr.

The tibiae were divided into four groups in which the defects were dealt with as follows:

Group I (n = 13). The defects were left empty with peripheral connective tissues, including the perichondrium and the zone of Ranvier, removed completely (Fig. 1 a).

Group II (n = 13). The defects were filled with grafts which retained their metaphyseal blood supply and consisted of hyaline cartilage from the peripheral margin of the secondary centre of ossification and the adjacent zone of Ranvier (Fig. 1 b).

Group llI (n = 7). The defects were filled with a non- vascularised periosteal flap taken from the proximal metaphysis immediately before implantation (Fig. 1 c).

Group IV (n = 7). As in group III, but with reattachment of the vascularised peripheral tissues, including the perichondrium and the zone of Ranvier (Fig. 1 d).

The grafts were anchored to the surrounding perichondrium with sutures in group II, or were held in place by the rapid regrowth of the overlying perichondrium in group III. In group IV, the grafts were covered by reattached peripheral connective tissues.

Before closing the wound, two parallel Kirschner wires were inserted into the epiphyseal and metaphyseal bone at a standard distance of 20 mm apart. An F-shaped template was used so that the wires were accurately placed in relation to each other in a valgus or varus direction. Transfixation of the growth plate was avoided and radiographs were taken to check the position. The wires marked the site of the defect and allowed measurement of longitudinal growth rate and angulation. As the wires were in the same place in all the animals, it was assumed that they were not responsible for any deformity.

A single intramuscular dose of streptomycin was given at the end of the operation. The animals were allowed to weightbear fully as soon as they had recovered. After 3 days in the animal house, they were transferred to a farm.

Anteroposterior and lateral radiographs, with a film focus distance of 80 cm, were taken immediately after the operation and before sacrifice.

Two sheep were killed immediately after the operation as controls for each group. These tibiae are not included in Table 1. The other animals were killed by an overdose of lethabarb at 2, 4, 6, 8, 12 and 24 weeks and the proximal tibiae removed. There were two tibiae at each date in groups I and II and one tibia in groups III and IV. The bones were fixed in 10% formalin containing 2% cetylpyridinium chloride and 5% Na2-EDTA for a maximum of two weeks. They were then

Table 1. Summary of the radiological and main histological findings in group I, II, III and IV tibiae

Group Procedure Growth rate Angulation Histological appearance

I (n = 12) Empty defect Normal No

II (n = 12) Normal No Hyaline epiphyseal cartilage of secondary centre of ossification + zone of Ranvier

III (n = 6) Free flap of periosteum Decrease Moderate

IV (n = 6) Free periosteum flap Severe Severe with reattached zone decrease of Ranvier

Defect filled with bone marrow-like tissue; no bone bridge.

Cavity filled as in group I; implant survived and continued to produce epiphyseal bone due to its original function; no growth plate repair

Gradual development of bone bridge formation which replaced connective tissues of the defect; osteoblasts adjacent to the periosteum

Earlier development of a large bone bridge formation in the the defect; disorganisation of epiphyseal plate adjacent to bone bridge


Longitudinal growth rate of group I-IV sheep

50

mm

48 Z 46 -- 44 ~ 42 ~ 40 -- 38 C 36 -~ 34 -- 32 ~ 30 - 28 - 26 C

24 ~ 22 C

20 - I ' I ' 1 ' 1 ' [ ' I ' 1 ' 1 ' 1 ' 1 ' [ ' I

2 4 6 8 10 12 1 4 16 18 2 0 2 2 2 4

W e e k s

' I ' I '

2 6 2 8 3 0

V a r u s angulation of group I-IV t ib iae

3 O

d 2 o

e g 1 5

r e lO e ~ S 5 4.

0 v / -

- 5 - - i i i i I i ~ i ~ i i i h h

2 4 6 8 10 12 14 16 18 20 22 24 26 28 3 0

weeks

b - - g r o u p I - - -4- g r o u p II ~ g r o u p I I I ~ g r o u p I V

Fig. 2. a Graph showing longitudinal growth rate in groups I to IV. (N = group I, • = group II, • = group III, A = group IV). b Graph showing varus angulation. (o -- group I, + = group II, * = group III, [] = group IV)

processed and embedded in paraffin. The specimens were cut in the coronal plane and 5 gm sections were stained with haematoxylin-eosin and alcian blue.

Results

Radiological assessment of longitudinal growth (Table 1)

The mean weekly longitudinal growth of group I over the 24 weeks was 1.10_+0.25 mm, and that of group II 1.07_+0.16 ram, which is within the normal growth rate for the limb [9]. In groups III and IV, slower growth occurred; the mean longitudinal growth rate in group III was 0.74__+0.22 ram/week compared with 0.59_+0.32 mm in group IV (Fig. 2a). There was no axial deformity in groups I and II, but in groups III and IV a varus deformity developed in addition to the slower growth rate (Fig. 2b). The deformity increased to 12 ° in group III and 28 ° in group IV by 24 weeks (Fig. 3). Radiographs at that time showed a marked bony bridge at the site of the original defect in group IV sheep.

Histological results (Table 1)

Group I. Immediately after the operation, the defect was filled with blood clot and fibrin which was replaced by granulation tissue and loose fibrous tissue at the end of two weeks. From the 4th to the 24th week, the fibrous tissue was uniformly replaced by bone marrow in which occasional haematopoetic precursor cells were found, although most of the defect contained loose adipose tissue. At the junction with the original growth plate, a small bony bridge was seen which led to

Fig. 3. Radiographs immediately after operation (a) and at 24 weeks in a group II (b) and a group IV (c) tibia with an obvious bony bridge and considerable angulation in the group IV tibia


Fig. 5. Photomicrograph (x2) of a tibia in group II after 12 weeks. The graft is at the site of its implantation (arrow- head) and is producing new bone; the defect (D) is filled with marrow-like tissue. The open arrow indicates the intact growth plate

Fig. 4. Photomicrograph (x44) of the junction between the defect (D) and the growth plate (G) showing the metaphyseal tether of the epiphyseal plate, a very small bony bridge (B) and degeneration of the growth plate cartilage in a tibia in group I at 6 weeks. M = metaphysis

metaphyseal deviation of the part of the growth plate next to the defect (Fig. 4). This bridge did not appear to affect longitudinal growth significantly as the growth rate (Fig. 2 a) was similar to previous measurements in normal limbs.

Group II. After implantation of hyaline epiphyseal cartilage and the adjacent zone of Ranvier, the postoperative haematoma was absorbed and replaced by loose fibrous connective tissue, and later by bone marrow, during the first 4 weeks after operation. As the whole bone grew in length, due to growth in the intact plate, the defect was noted to grow as well with the implant remaining at its original site at the bottom of the defect. However, the implant appeared to maintain its original function of bone growth (Fig. 5). The cellular structure of the epiphyseal cartilage was main- tained throughout the 24 weeks. The outer cell layer, representing the former perichondrium, was

still present as were the proliferating and hypertrophic chondrocytes of the inner layer which were undergoing endochondral ossification. At the medial side of the defect near to the growth plate, a small bony bridge with widening and deviation of the growth plate in a metaphyseal direction was seen. The bridge did not have a significant effect on longitudinal growth.

Group IlL The immediate postoperative findings were comparable with those already described. The flap of periosteum was embedded in a blood clot which was gradually resorbed and replaced by fibrous tissue within the first 2 weeks. By 8 weeks, new bone formation filled most of the defect bridging the epiphyseal and metaphyseal bone. The original periosteal flap could be identified and did not appear to have undergone any proliferation (Fig. 6 a). Active new bone formation was obvious next to the periosteum (Fig. 6b). New bone continued to be laid down and by 24 weeks most of the defect was replaced by a bony bridge.

Group IV. The histological appearances were similar to those in group III, although the formation of a bony bridge occurred earlier (Fig. 7 a).


Fig. 6. a Photomicrograph (x22) of a tibia in group III after 12 weeks showing a typical bony bridge. B = bony bridge; E = epiphysis; M = metaphysis; P = implanted periosteum, b Photomicrograph (x78) of a tibia in group IV after 8 weeks showing the osteoblasts (arrow) lined up around the periosteum. B = bony bridge; P = periosteal implant.

Fig. 7. a Photomicrograph (x22) of a tibia in group IV after 12 weeks. Endochondral ossification and growth are slower on the side of the bony bridge compared with the opposite side. b Photomicrograph (x78) is an insert of a. Structural abnormalities of the growth plate are seen adjacent to the defect in a tibia in group IV after 8 weeks

Osteoblasts were present on the edge of the tra- beculae near to the periosteum indicating osteogenic activity. Longitudinal growth decreased rapidly and there was no increase in the size of the defect after 6 weeks. The adjacent host growth plate showed disorganisation with loss of orderly chondrocyte columns and irregular ossification (Fig. 7 b), most pronounced near to the defect/bony bridge with normal structure present on the opposite side. After 12 and 24 weeks, the whole defect was replaced by new bone (Fig. 7a). The reattached zone of Ranvier on the outside of the epiphyseal plate remained intact throughout the 24 weeks.

Discussion

Fat, cartilage and biologically inert materials, such as silicone and methylmethacrylate [2, 10, 20, 28, 33, 44], have been implanted in clinical and experimental growth arrest and have led to satisfac- tory results by preventing the formation of bony bridges [3, 18, 21]. Nevertheless, experimental work has failed to show the restoration of the normal structure of the growth plate. Only fat has been reported to proliferate in the defect [22].

It is generally agreed that cartilage and bone are derived from pluripotent progenitor cells of mesenchymal origin. These stem cells are found in various tissues including bone marrow, muscle and periosteum in which the capacity for osteochon-


drogenesis has been demonstrated in vivo [25, 27, 31]. Variable extrinsic and intrinsic factors de- termine whether chondrogenesis or osteogenesis will take place [7].

The radiological results in our groups I and II confirm our previous findings that peripheral growth plate defects of less than 17.2 -t- 2.1% of the total physeal area of the ovine proximal tibia do not significantly affect the longitudinal growth rate [9]. This biological reaction is determined by the force of longitudinal growth provided by the intact plate [9].

The defects in group I are filled initially with fibrous tissue which is later replaced by tissue similar to normal bone marrow, although with fewer haematopoetic cells than normal; this tissue originates from the epiphyseal and metaphyseal marrow cavities. The reaction of the growth plate at the junction of the defect was consistent and a thin bony bridge was observed which could not be seen on radiographs. The plate was tethered to the bridge resulting in metaphyseal deviation as further growth occurred.

These results contrast with previous findings obtained after a similar procedure in rabbits. Bony bridge formation leading to partial growth arrest with significant deformity and leg length inequality was observed after excision of the lateral part of the plate and without the implantation of any material [23, 32, 33]. The critical size of growth plate defects in rabbits is not known and may have been exceeded in these experiments.

In group II the outcome was similar to group I, with the defect gradually filling with marrow-like tissue and a thin bridge formed producing distor- tion in a metaphyseal direction. The transposed cartilage remained in position presumably because of its distal attachment to the periosteum. Some new bone formation occurred in the area of the implant arising from the endochondral ossifying layers of the implant. Although this ossification proceeded in group II, it was not as rapid as in the adjacent growth plate leading to an increase in size of the defect. This reflects the normal rate of bone formation by the marginal ossifying cells. There was no evidence of transformation of the Ranvier cells into growth plate chondrocytes, or of their participation in repair other than as support tissue [3, 40].

Our findings emphasise the unique properties of the growth plate. Longitudinal growth arises from the high rate of reproduction of chondrocytes in the proliferative zone, combined with the increase in cell size during the hypertrophic phase of the cell life-cycle [17, 38]. The extracellular matrix sur-

rounding chondrocytes in the different regions of the plate is modified in preparation for mineralisation and the invasion of blood vessels [5, 6, 26].

The secondary centre of ossification also grows peripherally by endochondral ossification and this mechanism is similar to that of the growth plate with hypertrophy of cells and the appearance of type X collagen before mineralisation of the matrix [12]. This region is less organised than the growth plate with fewer proliferative cells and less well organised columns [15], and the hypertrophic region is less cellular. Our data show that these cells did not change their character on transplantation and that cell division rates are programmed early in life. Intrinsic growth plate chondrocytes alone possess the unique capacity for longitudinal growth [38].

The formation of a bony bridge is not only re- lated to the area of damaged growth plate [9], but also to the type of injury sustained [30, 34, 36]. Implantation of periosteum led to very significant bridge formation in groups III and IV. Growth plate damage in our group IV was very similar to clinical type 6 injury [30, 34, 36] which often leads to growth arrest and deformity. We believe that our findings represent experimental confirmation for these clinical observations. Periosteum possesses the potential to be either chondrogenic and/or osteogenic [27, 28, 29]. The physiological environ- ment of the growth plate area, with the presence of various growth factors, could well stimulate the mesenchymal stem cells to differentiate into osteoblasts resulting in bone deposition and bridge formation [14, 28]. Histologically, there was no proof of chondrogenic transformation of the periosteum in our specimens.

This study demonstrates that the functional, biochemical and physiological properties of different regions of the epiphysis of long bones in- volved in growth may be so specific that sub- stitution of one area for another does not alter the intrinsic nature of the tissues. Transplantation of hyaline cartilage of the secondary centre of ossification and of the zone of Ranvier survives and persists as cartilaginous tissue, but is unable to restore, repair or function as a growth plate in these conditions. No significant recruitment of cells to provide longitudinal growth has been observed. It is most likely that bony bridge formation after implantation of periosteum is supported by the osteogenic properties of this tissue. Further experiments may need the use of growth factors to promote specific stimulation in an attempt to in- duce this tissue to repair defects, other than acting as another biological interposition material.


Acknowledgements. The authors would like to express their gratitude to Mr Ray Yates and his staff for taking care of the animals at Flinders Medical Centre/University Laboratory and for their help during the experiments. We would also like to thank Mrs Sue Teller (Department of Histopathology) for her assistance with the histological sections. Mr Roland Hermanis (Department of Histopathology) is thanked for his support with the photography of the histological sections.

This work was supported by grant no: 880550 from the National Health and Medical Research Council of Australia and the Adelaide Bone and Joint Research Foundation.

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Documents

The implantation of cartilaginous and periosteal tissue into growth plate defects