Annals of the Royal College of Surgeons of England (1975) vol 57
Articular cartilage studies
and
osteoarthrosis George Bentley chM FRCS Clinical Reader, Nuffield Department of Orthopaedic Surgery, University of Oxford
Summary Osteoarthrosis is characterized in the early stages by degradation of articular cartilage matrix. Clinical, radiological, and pathological studies have failed to reveal the factors which initiate the breakdown of cartilage and are not applicable to detailed sequential studies of the affected tissues at all stages in the disease. Therefore animal experiments have been employed to provide more information on degradation and repair processes in cartilage. These studies have demonstrated: i) Matrix protection and induced repair of mature articular cartilage by the use of oral aspirin after lacerative injury. 2) Establishment by the intra-articular injection of the plant enzyme papain of a model of osteoarthrosis in the rabbit hip which mimics human osteoarthrosis and is suitable for further experimental studies. 3) A proliferation of mature articular cartilage chondrocytes in response to loss of matrix, which indicates a latent repair capacity. 4) Repair of extensively damaged hip joints after femoral osteotomy by increased formation of subchondral new bone and formation of fibrocartilage on the articulating surfaces. These tissue repair processes are associated with an increase in vascularity of the femoral head and acetabulum produced by the osteotomy. 5) Successful transplantation as allografts in both normal and arthrotic rabbit knees
of aggregates of epiphysial chondrocytes isolated from their matrix. This method of joint surface replacement may have clinical applications.
Introduction Osteoarthrosis is a cause of extensive crippling due to involvement of the major weightbearing joints such as the hip, the knee, and the spine, and several surveys have indicated that approximately 30% of the population over the age of 35 years have involvement of one or more joints with osteoarthrosis" 2. John Hunter was aware of the generalized nature of the disease and of the extent of involvement of joints in the population as indicated by Figure i, which shows specimens from the Hunterian Collection. These include a cervical spine (Specimen P.953) affected by osteoarthrosis with osteophytes on the margins of the vertebral bodies and posterior spinal joints, a humerus (Specimen P.959) with erosion and eburnation of the articular surface and formation of large marginal osteophytes, and a knee joint (Specimen P.96o) with extensive involvement by osteoarthrosis in which Hunter described ebumation of the articular surfaces, osteophyte formation, and the formation of loose bodies from the articular surfaces3. The patella (Specimen P.96i), which was taken from a man who 'had bruised the knee and died shortly afterwards', shows fissuring and fibrillation of the
From a Hunterian Lccture delivered on 8th May I974
Articular cartilage studies and osteoarthrosis
87
FIG. I Specimens from the Hunterian Collection showing osteoarthrosis of different joints (see text). Reproduced by kind permission of the Curator of the Hunterian Collection, Royal College of Surgeons of England.
undersurface and is remarkable in that it is the first specimen to show traumatic chondromalacia of the patella. By the beginning of the 2oth century it was realized that articular cartilage had a unique structure. It was then known to have no
blood vessels, no lymphatics, and no nerve supply and to have almost no capacity for repair after injury. However, in the past 20 years radioisotope studies have demonstrated that the articular cartilage chondrocytes are as active as other connective tissue cells in
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George Bentley
turning over the components of the imfatrix. The reason, therefore, for the apparient inability of cartilage to heal after injutry lies not in a defect of cells but in its unique structure and in particular the low cell-tomatrix ratio. Articular cartilage (Fig. 2) iS compc)sed of four zones: a superficial (Zone I), ant intermediate (Zone II), and a radial (Zoi III) separated from the deep, calcified Zc)ne IV by the 'tide-mark', which is a calcification front. The arrangement of collagen filbres in cartilage is in a criss-cross pattern wiith the deep collagen fibres embedded in the c;alcified zone and the superficial fibres blendin g with an outer limiting membrane of fine fibres 3 ,um thick named the 'lamina sple: ndens'. The collagen pattern is designed to withstand the compression and shearing stresses involved in joint load-bearing.
ie
FIG. 3 Diagram to illustrate how the osmotic effect of matrix proteoglycan (Pg) maintains the open collagen meshwork of articular cartilage.
Between the meshes of the collagen fibres lie the chondrocytes which secrete the collagen and also the proteoglycans which form the packing substance of the matrix. Proteoglycan is composed of a protein core with approximately 50 side-chains of glycosaminoglycans, the commonest of which are chondroitin-4-sulphate and chondroitin-6-sulphate. The chondroitin sulphate carries a negative charge which attracts water into the tissue and holds it by osmotic pressure. As a result 70% of articular cartilage is composed of water. Thus the collagen meshwork is held tense by the osmotic pressure of the proteoglycan and hence cartilage is resilient in the normal situation (Fig. 3). If there is loss of glycosaminoglycans from the cartilage, then there is loss of water and hence resiliency, and cartilage breakdown with fracture of the collagen fibres, which is seen histologically as fibrillation, will occur. IV It is important to note that the chondrocytes of articular cartilage are not inert, and on electron microscopic examination they show an extensive endoplasmic reticulum and FIG. 2 Normal mature articular cazrtilage Golgi apparatus, indicating that they are proshowing arrangement of cells and matrix ducing protein and glycosaminoglycans. The in 4 zones. synthetic functions of chrondrocytes are:
Articular cartilage studies and osteoarthrosis (I) synthesis of protein for proteoglycans, collagen, and new cells; (2) synthesis and polymerization of glycosaminoglycans; and (3) sulphation of glycosaminoglycans. In osteoarthrosis the first observed histological abnormality is loss of matrix from the upper Zone II of the articular cartilage. This is followed by chondrocyte death, chondrocyte cluster formation, fibrillation of the cartilage, and erosion. Secondary changes -of thickening or ebumation, marginal osteophytes, and cysts-with alterations in blood flow which may be hyperaemia or venous stasis in different areas, occur in the underlying bone. The result of these changes is deformation of the surfaces of the joints and feeble repair response in the form of tufts of granulation tissue arising from the subchondral marrow. The mechanism of breakdown of articular cartilage is demonstrated schematically in Figure 4. Under the influence of compression-shearing stresses there is damage to cells in the upper Zone II of the cartilage. This results in the release of their catheptic enzymes, particularly
COMPRESSION SHEARING STRESSES
CELL DAMAGE mLY
MiNGLYCO$AIKOGLYCANS N.',;AND WATER LOSS
LOSS OF
CARTILAGE
RESILIENCY
FISSURES
CATIHEPSIN RELEASE
FURTHER CELL DAMAGE
L
LYSOMAL STABILISING AGENTS
RELEASE
Cartilage breakdown sequence in ostleoarthrosis, showing possible effect of lysosomal stabilizing agents. FIG. 4
89
cathepsin D, which are contained in the lysosomes of the cells. The cathepsin breaks the bond between the protein core and the glycosaminoglycans of the neighbouring matrix, resulting in loss of glycosaminoglycans. Hence there is water loss and loss of resiliency of the cartilage. As a result fissures occur. In the areas around the fissures further cells are subjected to excessive stress and are damaged. More enzymes are released from the lysosomes of these cells which digest more matrix; thus a vicious circle of breakdown of cartilage is produced and hence erosion of the articular cartilage occurs. The weight-bearing stress and movement of the joint surfaces cause a progressive breakdown of the articular cartilage. In response to the injury two processes occur in articular cartilage: (i) feeble chondrocyte proliferation and (2) increased synthesis of glycosaminoglycans. It is these two responses that I will consider first.
Repair of injuries limited to articular cartilage Mankin and Boyle4 have shown that when articular cartilage alone is injured by laceration of the surface there is a response in immature cartilage of proliferation of chondrocytes. This has been demonstrated by labelling the cartilage with tritiated thymidine, which is taken up in the S phase of mitosis. They found a mitotic index of i6o per I00 000 cells in newborn animals, but by the age of 6 months the mitosis in cartilage in response to injury had almost completely disappeared. Thus it was established that adult articular cartilage cells have almost no capacity for replication even after injury. However, Ash and Francis5 have demonstrated that when adult articular cartilage chondrocytes are isolated from their matrix and exposed to liver somatomedin in tissue culture the uptake of tritiated thymidine is
go
George Bentley
greatly increased, which implies a greatly increased cell division rate. However, the healing of articular cartilage requires the formation of new matrix as well as cell division. In I965 Simmons and Chrisman' demonstrated that rabbits which were fed with sodium salicylate in therapeutic doses showed diminished breakdown of the articular cartilage of the knee after laceration. Also, by 8 weeks from the time of the laceration, in some animals healing of the cartilage was occurring which was not observed in controls. It was thought that the apparent healing in Simmons and Chrisman's experiment could have been related to the direction of the lacerations in the cartilage, since in different areas of joints the collagen fibre orientation in the superficial zone is such that it resists the major stresses. Thus it appeared that if the lacerations in cartilage were made in the direction of the predominant orientation of the collagen they would be more likely to heal than if they were made across the line of orientation. As a result a preliminary experiment was carried out on 24 mature rabbits. Twelve received lacerations in the femoral head articular cartilage in a radial direction (in line with the collagen fibres) and I 2 in a transverse direction. It was found that there was no healing of the cartilage in either group. A second experiment was performed on I4 animals, 7 of which received acetylsalicylic acid in their feed to produce a serum salicylate level of 1.09- .82 mmol/l (I 5-25 mg/ ioo ml). In this preliminary study examination of the cartilage sections showed that in those animals which had received a therapeutic dose of acetylsalicylic acid for 8 weeks healing of cuts had occurred (Fig. 5), whereas in the control animals it had not. These results suggest that acetylsalicylic acid has a healing effect on articular cartilage which
may be achieved either by diminishing breakdown of matrix due to lysosomal stabilization, thus allowing repair, or alternatively by stimulating matrix formation in some way. As a result of these investigations I have adopted the practice of treating all patients who have undergone arthrotomy of joints with possible cartilage damage-for example, those who have undergone meniscectomywith acetylsalicylic acid in therapeutic dosage for 8 weeks after operation. This is in an attempt to reduce the incidence of osteoarthrosis of the knee in patients who have undergone meniscectomy, which is known to be 25% over a period of 25 years7. It is im-
femoral head showing healing Of 3 lacerations after 8 weeks of oral. aspirin. (Toluidine blue, X 25.) mature rabbit
Articular cartilage studies and osteoarthrosis portant to realize that many other drugs have lysosomal-stabilizing properties-for example, glucocorticoids, chloroquine, inorganic gold salts, indomethacin, and phenylbutazone. Investigation of the effects of these drugs on articular cartilage is of great importance when considering the early prevention of cartilage breakdown and therefore of osteoarthrosis.
9I
papain to produce selective matrix breakdown appeared logical as a method of producing osteoarthrosis since the mechanism of its action is to break the bond of matrix proteoglycans between the protein core and the glycosaminoglycans of the side-chain in a manner comparable to that produced by cathepsin D (Fig. 6). This enzyme was employed, therefore, in an attempt to produce an experimental osteoarthrosis in mature
Experimental osteoarthrosis It is important for further study of the patho- animals8. Injections of 0.2 ml of 2% sterile papain genesis of osteoarthrosis and its response to various methods of treatment to observe solution activated by o.i ml of 0.03 M cystchanges at all stages in the development of eine solution were made into the hip joints the disease. Investigations into the effects of 40 mature Chinchilla rabbits on the ist, of different methods of treatment on joints 4th, and 7th days. The opposite hips received damaged by osteoarthrosis is restricted in the 3 injections of isotonic saline in the same living patient by the inaccessibility of the disx + eased tissues. Therefore there is a need for a model of osteoarthrosis which can be reproduced consistently in animals, which is recognizable without sacrifice of the animals, and which is produced in a manner similar to that occurring in human arthrosis. Table I shows the methods of inducing experimental osteoarthrosis which have been employed to date. All of these methods produce lesions which are not similar to osteoarthrosis or produce them by an effect which is not comparable to the mechanism of breakdown of articular cartilage in osteoarthrosis. The use of the proteolytic plant enzyme TABLE I Methods used for inducing experimental osteoarthrosis Lacerations, excision, percussion, and cautery Chemical (acids, alkalis) Foreign bodies (talc, cartilage fragments) Prolonged immobilization and compression Subluxations, dislocations, and relief of contact Hormones (growth, sex, steroids) Venous stasis Genetic Dietary fat
FIG. 6 Diagrammatic representation of action of papain and cathepsin D on proteoglycan molecule.
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George Bentley
volume as the right hip, as controls. The changes produced in the hip joints were studied at intervals from 6 h to 9 months by radiography, photography, and histological examination after staining with haematoxylin and eosin and with periodicacid-Schiff (PAS) counterstained with alcian blue to show the distribution of glycosaminoglycans.
Findings 6 h after one papain injection No radiographic changes were visible and the femoral head and acetabulum appeared normal to the naked eye. The articular cartilage over the superior surface of the femoral head and opposing part of the acetabulum was softer than on the control side when pressed with a blunt probe. Histologically, the cartilage of the upper surface of the femoral head showed slight fibrillation and indentation. There was loss of glycosaminoglycan staining of the matrix and also loss of nuclear staining of the chondrocytes, indicating their death, in Zones I and II (Fig. 7). The nuclei of some of the deeper chondrocytes were pyknotic. The acetabulum showed similar but less advanced changes. The changes in the articular cartilage at this stage resembled those observed in early
the central area-that is, Zones I-IIL-at the periphery the deep cells were more extensively affected than the superficial ones. The upper acetabular changes were more advanced than those at one day, with loss of matrix staining and chondrocyte death, occasional chondrocyte cluster formation, indentation of the surface articular cartilage, and the beginning of horizontal cleft formation. Findings 6 weeks after last papain The radiographs at this stage injection showed obvious narrowing of the joint space in the right hip, with slight sclerosis of the subchondral bone of the femur and acetabulum.
osteoarthrosis9"l0. Findings one week after last papain On naked-eye examination injection more widespread fibrillation of the articular surface of the upper femur was seen, with similar changes in the acetabulum. Histologically, loss of cartilage matrix staining was observed on the upper surface of the femoral head, with more extensive fibrillation of the articular cartilage than that seen after 6 h. Although the chondrocytes were dead throughout the thickness of the articular cartilage as far as the calcified layer in
FIG. 7 Section of articular cartilage of femoral head of mature rabbit 6 h after
intra-articular injection of 2% papain solution. (PAS and alcian blue, X 25.)
Articular cartilage studies and osteoarthrosis
93
was joint-space narrowing, sclerosis of subchondral bone of the femur and acetabulum, and subluxation of the femoral head. Small osteophytes were seen on the upper margin of the femoral head (Fig. 8). Macroscopic examination revealed extensive denuding of articular cartilage down to subchondral bone and similar though slightly less extensive damage to the acetabulum. Marked thickening of the joint capsule was present. The photomicrographs showed complete loss of articular cartilage down to subchondral bone in the weight-bearing areas. The subchondral bone was thickened and there was relative osteoporosis towards the margin of the femoral head; occasional subchondral cysts were seen (Fig. 9). Osteophytes were seen on the superior margin of the femoral head, with vessels and bone growing into the base of the articular cartilage. The acetabulum showed similar denuding of articular cartilage with underlying thickened subchondral bone. It is important to note that there was no sign of replacement of the surface of the femoral head or acetabulum by the remaining articular cartilage or from subchondral bone cells. At this stage the synovial membrane was Findings 9 months after last papa'n slightly inflamed and there were fragments injection The radiographic appear- of cartilage and bone engulfed and lying beances were advanced to the stage where there neath the surface. The femoral head showed flaking, fibrillation, and erosion of articular cartilage over the superior surface, as did the opposing acetabulum. Histologically, it was at this stage that the secondary response of the subchondral bone to the articular cartilage damage was first seen. The photomicrographs showed fibrillation and erosion of the articular cartilage on the upper surface of the femoral head extending into Zone III, but some of the deep chondrocytes remained alive. The most notable feature was the presence of new blood vessels invading the base of the articular cartilage at the periphery of the femoral head, with the formation of new bone. This process of endochondral ossification is an appearance very similar to that seen in human osteoarthrosis'0, and was the earliest manifestation of osteophyte formation in the experiment. The acetabulum showed similar but less advanced changes to the femoral head. The synovial membrane showed hyperplasia of the surface cells, and large fragments of articular cartilage were seen engulfed within it. The appearances were comparable to those seen in the synovium of osteoarthrotic joints'1.
FIG. 8 Radiograph of hips of mature rabbit 9months
after 3 injections of pap-
jJ
amn into right hip (see
~~~~text).
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George Bentley
FIG. 9 Section of femoral head 9 months after 3 in-
jections of papain (see text).
Table II shows the changes produced in papain arthrosis. It will be seen that the changes correspond to those seen in human osteoarthrosis in most features. The sequence of changes produced in this experiment demonstrate that advanced changes similar to those of human osteoarthrosis can develop in previously normal rabbit joints from primary changes induced in the articu-
lar cartilage matrix. This supports the view that osteoarthrosis developing in a previously normal joint is due to primary degeneration of articular cartilage rather than to changes in the subchondral bone.
Chondrocyte cluster formation in mature articular cartilage It has been noted that chondrocyte dcusters
TABLE II Changes produced in papain arthrosis compared with those in osteoarthrosis Papain arthrosis Osteoarthrosis Articular cartilage
Glycosaminoglycan loss Chondrocyte death and clumping Fibrillation Fissuring Erosion Subchondral bone Sclerosis Osteophytes Cysts Synovium Hyperplasia Cartilage and bone debris Increased vascularity Subsynovial fibrosis
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes
Yes Yes Occasional
Yes Yes
Yes Yes Yes Yes
Yes Yes Yes Yes
Yes
Articular cartilage studies and osteoarthrosis were seen in the articular cartilage within the first 24 h after the injection of papain. It has been argued on the one hand that they form as a result of multiplication of cells in response to damage of the cartilage and on the other that they represent passive aggregation of dead or dying cells secondary to loss of cartilage matrix. An experiment was carried out in which one injection of 2% sterile papain solution with 0.03 M cysteine as activator was made into the right hip joint of 4 mature New Zealand white rabbits. Isotonic saline was injected into the opposite hip as control. Rah bits were killed after 6 h and 24 h and the femoral heads and acetabula of both hips were incubated in a nutrient medium containing tritiated thymidine in a concentratinon of ioo mCi/l. Autoradiographs were prepared of serial sections of the femoral
95
heads and acetabula by the dipping technique of Joftes'2. It was found that chondrocyte clusters occurred in particular in the acetabula of the experimental hips after 6 and 24 h. Only occasional clusters were seen in the femoral heads. The counts from the serial sections were expressed as mitotic indices. The mitotic index of articular cartilage was increased in the papain-injected hip and to a slight extent in the opposite hip, presumably owing to a systemic effect of the papain. The number of mitoses fell after 24 h, suggesting that cartilage cell division was short-lived after the loss of matrix produced by one papain injection (Fig. io). It was noted that 5O% of the cells labelled in the autoradiographs were within chondrocyte clusters. This appeared to confirm that chondrocyte cluster formation was due to proliferation of articular cartilage cells. F
=
Ac.
Femur =
Acetabulum
* Experiment
Ac.R +
/
30-
Control
/
20*
Ac.R
10
/Ac
MITOTIC INDEX No. of Mitoses
12
/100,000 cells
1'2
6 TIME FROM INJECTION
-
AcL 1.
1824
HOURS
FIG. I0 Mitotic index of mature femoral head articular cartilage after one injection of papain into the hip.
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George Bentley
Repair capacity of arthrotic joints Next I would like to consider the response of joints which have lost articular cartilage, thus exposing the sibchondral bone, but which are not grossly distorted in their architecture so that clinically they are painful but still mobile. This is the stage at which many patients present for treatment, and the microscopic features correspond to those seen 8 weeks after 3 papain injections into a rab bit hip. The healing effect of intertrochanteric osteotomy of the arthrotic hip joint is well known but poorly understood. It has been observed that after femoral osteotomy bone cysts and sclerosis disappear from the femoral head on the radiographs, and the formation of a new joint space is observed in approximately 6o% of cases'3'14. It was decided to investigate these observed clinical phenomena experimentally. Two months after the induction of papain arthrosis in the hip joint I4 mature Chinchilla rabbits underwent high femoral osteotomy of the femur of a non-displacement type which was fixed with an intramedullary screw. This was to distinguish the biological effects of the osteotomy from any mechanical effects. Fourteen animals with papain arthrosis of the hips which had been induced at the same time were the controls. The animals in each group were divided into two subgroups, and after 3 months and 6 months respectively microangiography of the hip joints was performed by the intra-aortic injection of Colorpaque at a pressure of ioo mm Hg (I3.3 kPa). The animals were then killed and the femoral heads and acetabula examined histologically. The femoral osteotomies were united within 6 weeks of operation. Findings 3 months after femoral osteoIn 5 out of 7 animals which tomy had undergone femoral osteotomy micro-
FIG. II Microangiograms of femur: (a) 3 months after femoral osteotomy; (b) with papain arthrosis only.
angiography showed an increase in the number of arterioles in the femoral head and acetabulum compared with the controls (Fig. iI). No such increase was observed in the controls in comparison with normal hips. Thus the increase of arterioles was due to the osteotomy. Examination of the histological sections showed a consistent increase in the thickness of the subchondral bone plate of the femoral head in the osteotomy group, indicating increased formation of new bone. Findings 6 months after femoral osteoIn 6 out of 7 animals which tomy had undergone femoral osteotomy there was an increase in the number of arterioles in the femoral head and acetabulum despite the fact that the osteotomies were united by 6 weeks. Histologically, at this stage two types of surface repair of the denuded bone of the femoral head and acetabulum were seen. Those areas with small defects showed filling with fibrous tissue which was clearly emerging from the subchondral bone and undergoing cartilage metaplasia. Where there
Articular cartilage studies and osteoarthrosis
97
FIG. 12 Acetabulum from hip with papain arthrosis 6 months after femoral osteotomy. The surface is denuded of articular cartilage but 3 tufts of fibrocartilage are seen emerging from the subchondral marrow. (PAS and alcian blue, X io.)
Biological replacement of articular surfaces by transplantation of isolated chondrocytes Many joints affected by osteoarthrosis are damaged beyond the stage where induction of repair by the use of lysosomal-stabilizing drugs or juxta-articular osteotomy are of value. As a result many attempts have been made over the past 70 years to replace articular surfaces with grafts of articular cartilage It is thus possible to explain the pheno- and a thin layer of subchondral bone in both mena following femoral osteotomy in man as indicated in Figure I 3. The effect is augMECHANICAL EFFECT mented by the more favourable distribution BIOLOGICAL 4 EFFECT of stresses through the damaged area of the Increased vascularity I Redistribution of stresses joint after displacement osteotomy. 4 4 Increased New bone marrow The importance of these observations lies formation activity I in the possibility of inducing the changes of . Fibrocartilaqe I of hyperaemia, increased subchondral bone for- Clearinq formation cysts and mation, and joint-space repair by non- sclerosis 4 Joint healing to a lead could This means. operative l Pain relief of inmethods the of widening application ducing repair to other joints, where juxta- FIG. I3 Effect of femoral osteotomy on osteoarthrotic hip. articular osteotomy is not applicable.
were larger areas of denuding of the articular surface down to bone intense activity of the subchondral marrow cells was seen. Arising from these cells were tufts of fibrocartilage seen on the surface of the femoral heads and acetabula (Fig. I2). An extension of this fibrocartilage formation could have led to the formation of a continuous surface layer.
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humans and experimental animals'". There are two problems with such osteochondral allografts derived from, for example, cadavers. They are: (i) fixation of the grafts at the host site and (2) rejection of the graft by the host. A new approach to biological replacement of joints is therefore indicated. In I968 Chesterman and Smith"' grafted articular cartilage chondrocytes of adult rabbits, which had been isolated from their matrix by enzymatic digestion with papain and collagenese, into a defect cut in the upper articular surface of the humerus of adult rabbits. These allografts failed to survive. I decided to employ young cells taken from 6-week-old New Zealand white rabbits, since their capacity for cell division and matrix production is much greater than that of mature cells". The cells were isolated from the epiphysial growth plate of the metatarsals and also from the articular cartilage of the knee by digestion of the matrix with trypsin and collagenase and then, after gentle centrifugation at 5oo/min, transplanted into holes 3 mm wide and 3 mm deep drilled in the tibial articular surfaces of knee joints of adult rabbits of the same species. Transplantation into normal joints In the initial experiment the epiphysial cells and articular cartilage cells were each implanted into 8 mature rabbits. The controls were identical drill holes in the opposite knee which did not receive cells. The animals were killed after 8 weeks. The tibial condyles containing the grafts were incubated in nutrient medium containing IOO LCi of "5S-sulphate for 3 h at 370C and autoradiographs were then prepared. Histological sections 5 ,um thick were stained with alcoholic toluidine blue. Articular cartilage allografts Eight weeks after implantation of articular cartilage cells the defects appeared to be
FIG. 14 Allograft of immature articular cartilage 8 weeks after implantation. Graft is surrounded by inflammatory cells and fibrous tissue and is not incorporated. (Toluidine blue, X 7.5.)
filled with cartilage tissue in 2 of the 8 animals. Histologically, examination revealed that although there was cartilage present in 2 of the 8 defects it was not being incorporated, and intense inflammatory reaction was occurring, suggesting rejection of the grafted cells (Fig. I 4). Epiphysial cartilage allografts Eight weeks after implantation of epiphysial cartilage cells into the articular surfaces of rabbit tibae there was filling of the defects almost completely in 6 of the 8 animals. Histologically, the appearances were notable. There was filling of the defects with cartilage which was incorporated and which showed normal metachromasia of the matrix with toluidine blue staining (Fig. I5). In addition the autoradiographs demonstrated a normal distribution of 35S-sulphate, indicating that these cells were viable. The control drill holes showed only fibrous tissue loosely filling the
Articular cartilage studies and osteoarthrosis
99
physial and articular chondrocytes were implanted into drill holes in exactly the same way as in the normal joints and the animals were killed after 8 weeks. It was found that of the 8 joints which had received epiphysial cells complete filling of the defects had occurred in 3 and partial filling of the defect in a further 3. Of the 6 joints which had received articular cartilage cells partial cartilage filling had occurred in 2, but the remaining 4 showed only fibrous tissue in the defects FIG. I 5 Allograft of epiphysial cartilage 8 (Fig. I6). weeks after implantation. Cartilage fills the defect and is incorporated into host cartilage Conclusions It is concluded that the employment of epiand bone. (Toluidine blue, X 7.5.) physial cartilage chondrocytes as allografts for the filling of defects in articular surfaces may defects. There was no sign of rejection of have a clinical application, particularly if a the grafts. method of long-term storage of cells by deepproves possible. The reason for the survival of the epiphysial freezing that future biological reproposed It is cartilage cells, which has been confirmed in search into articular cartilage and osteofurther experiments, is not certain, but it may be because of their greater activity than articular cells for cell division and matrix production. Heyner'8 demonstrated that isolated chondrocytes can produce new matrix within io h of isolation from their original matrix and it is also known that the host antibody reaction requires approximately one week to become fully developed. It is proposed that epiphysial cartilage cells, because of their greater capacity for producing matrix compared with articular cells, can produce matrix rapidly enough to protect themselves from rejection by the host antigen-antibody reaction. Transplantation into joints with papain arthrosis The clinical application of this method of grafting is likely only if isolated chondrocytes can survive in an arthrotic joint. FIG. I 6 Allograft of epiphysial cartilage 8 Accordingly, in a small preliminary experi- weeks after implantation in knee with papain ment papain arthrosis was induced in the arthrosis. Healthy cartilage fills defect. (Toluknee joints of I4 mature rabbits. Isolated epi- idine blue, X 7.5.)
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George Bentley
arthrosis should be developed in five fields: i) The early diagnosis of osteoarthrosis in patients before radiological changes become apparent. 2) Prevention by lysosomal-stabilizing drugs of cartilage breakdown in patients whose joints are at risk from previous injury. 3) The production by growth hormone or somatomedin of repair in early cartilage damage. 4) Induction of fibrocartilage surface repair in damaged joints by osteotomy, surface drilling, or non-operative means such as hyperbasic oxygen therapy. 5) Replacement of grossly damaged surfaces by a technique of multiple drillings together with cartilage cell allografts. Although a cure is not yet available for ostcoarthrosis, pursuance of the avenues of research considered in this lecture should reduce the incidence of degeneration of articular cartilage and osteoarthrosis in those particularly at risk and reduce the necessity for extensive and sometimes difficult late reconstructive surgery in the future. I have pleasure in acknowledging the help given to me by Robert B Duthie, Nuffield Professor of Orthopaedic Surgery, University of Oxford, in whose department the above studies commenced anid finished. Professor A B Ferguson jr, of the University of Pittsburgh, generously provided a scholarship for one year. Technical help was provided by D W HIaynes, Mrs H Gorgescu, and Mrs I)ianne Deane. The illustrations were prepared by the photographic department of the Nuffield Orthopaedic Centre, and the manuscript was typed by Mrs Janet Lever.
References I
Heine, J (1926) Virchows Archiv fur pathologische Anatomie und Physiologie und fur klinische Medizin, 260, 52I.
I)anielsson, L G (I964) Acta orthopaedica Scandinavica, Suppl. 66. 3 Hunter J (1759) from John Hunter's manuscript (54) in the possession of the Royal Collegc of Surgeons of England. 4 Mankin, H J, and Boyle, C J (I967) Cartilage Degradation and Repair, p I85. Washington, D.C., National Academy of Sciences. 5 Ash, P, and Francis, M J (I974) Personal communication. 2
6 Simmons, D P, and Chrisman, 0 D (i 965) Arthritis and Rheumatism, 8, 960.
7 Jackson, J P (I967) Journal of Bone and Joint Surgery, 49B, 584. 8 Bentley, G (I97I) Journal of Bone and Joint Surgery, 53B, 324. 9 Collins, D H, and McElligott, T F (I960) Annals of the Rheumatic Diseases, 19, 3I8. IO
Harrison, M H M, Schajowicz, F, and Trueta, J (I953) Journal of Bone and Joint Surgery,
35B, 598. ii
Lloyd-Roberts, G C (1953) Journal of Bone and Joint Surgery, 35B, 627.
12
Joftes, D L (I959) Journal of Laboratory Inzvestigation, 8, I31.
13 Duthie, R B, and Howe, W W (I963) Clinical Orthopaedics and Related Research, 3I, 65.
14 Ferguson, A B jr (I964) Journal of Bone and Joint Surgery, 46A, II59.
I5 Bentley, G (I972) ChM Thesis, University of Sheffield. i6 Chesterman, P J, and Smith, A U (I968) Journal of Bone and Joint Surgery, 5oB, i84.
I7 Mankin, H J (I968) Bulletin of the Newv York Academy of Medicine, 44, 545. i8 Heyner, S (I969) Transplantation, 8, 666.
Articular cartilage studies
and
osteoarthrosis George Bentley chM FRCS Clinical Reader, Nuffield Department of Orthopaedic Surgery, University of Oxford
Summary Osteoarthrosis is characterized in the early stages by degradation of articular cartilage matrix. Clinical, radiological, and pathological studies have failed to reveal the factors which initiate the breakdown of cartilage and are not applicable to detailed sequential studies of the affected tissues at all stages in the disease. Therefore animal experiments have been employed to provide more information on degradation and repair processes in cartilage. These studies have demonstrated: i) Matrix protection and induced repair of mature articular cartilage by the use of oral aspirin after lacerative injury. 2) Establishment by the intra-articular injection of the plant enzyme papain of a model of osteoarthrosis in the rabbit hip which mimics human osteoarthrosis and is suitable for further experimental studies. 3) A proliferation of mature articular cartilage chondrocytes in response to loss of matrix, which indicates a latent repair capacity. 4) Repair of extensively damaged hip joints after femoral osteotomy by increased formation of subchondral new bone and formation of fibrocartilage on the articulating surfaces. These tissue repair processes are associated with an increase in vascularity of the femoral head and acetabulum produced by the osteotomy. 5) Successful transplantation as allografts in both normal and arthrotic rabbit knees
of aggregates of epiphysial chondrocytes isolated from their matrix. This method of joint surface replacement may have clinical applications.
Introduction Osteoarthrosis is a cause of extensive crippling due to involvement of the major weightbearing joints such as the hip, the knee, and the spine, and several surveys have indicated that approximately 30% of the population over the age of 35 years have involvement of one or more joints with osteoarthrosis" 2. John Hunter was aware of the generalized nature of the disease and of the extent of involvement of joints in the population as indicated by Figure i, which shows specimens from the Hunterian Collection. These include a cervical spine (Specimen P.953) affected by osteoarthrosis with osteophytes on the margins of the vertebral bodies and posterior spinal joints, a humerus (Specimen P.959) with erosion and eburnation of the articular surface and formation of large marginal osteophytes, and a knee joint (Specimen P.96o) with extensive involvement by osteoarthrosis in which Hunter described ebumation of the articular surfaces, osteophyte formation, and the formation of loose bodies from the articular surfaces3. The patella (Specimen P.96i), which was taken from a man who 'had bruised the knee and died shortly afterwards', shows fissuring and fibrillation of the
From a Hunterian Lccture delivered on 8th May I974
Articular cartilage studies and osteoarthrosis
87
FIG. I Specimens from the Hunterian Collection showing osteoarthrosis of different joints (see text). Reproduced by kind permission of the Curator of the Hunterian Collection, Royal College of Surgeons of England.
undersurface and is remarkable in that it is the first specimen to show traumatic chondromalacia of the patella. By the beginning of the 2oth century it was realized that articular cartilage had a unique structure. It was then known to have no
blood vessels, no lymphatics, and no nerve supply and to have almost no capacity for repair after injury. However, in the past 20 years radioisotope studies have demonstrated that the articular cartilage chondrocytes are as active as other connective tissue cells in
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George Bentley
turning over the components of the imfatrix. The reason, therefore, for the apparient inability of cartilage to heal after injutry lies not in a defect of cells but in its unique structure and in particular the low cell-tomatrix ratio. Articular cartilage (Fig. 2) iS compc)sed of four zones: a superficial (Zone I), ant intermediate (Zone II), and a radial (Zoi III) separated from the deep, calcified Zc)ne IV by the 'tide-mark', which is a calcification front. The arrangement of collagen filbres in cartilage is in a criss-cross pattern wiith the deep collagen fibres embedded in the c;alcified zone and the superficial fibres blendin g with an outer limiting membrane of fine fibres 3 ,um thick named the 'lamina sple: ndens'. The collagen pattern is designed to withstand the compression and shearing stresses involved in joint load-bearing.
ie
FIG. 3 Diagram to illustrate how the osmotic effect of matrix proteoglycan (Pg) maintains the open collagen meshwork of articular cartilage.
Between the meshes of the collagen fibres lie the chondrocytes which secrete the collagen and also the proteoglycans which form the packing substance of the matrix. Proteoglycan is composed of a protein core with approximately 50 side-chains of glycosaminoglycans, the commonest of which are chondroitin-4-sulphate and chondroitin-6-sulphate. The chondroitin sulphate carries a negative charge which attracts water into the tissue and holds it by osmotic pressure. As a result 70% of articular cartilage is composed of water. Thus the collagen meshwork is held tense by the osmotic pressure of the proteoglycan and hence cartilage is resilient in the normal situation (Fig. 3). If there is loss of glycosaminoglycans from the cartilage, then there is loss of water and hence resiliency, and cartilage breakdown with fracture of the collagen fibres, which is seen histologically as fibrillation, will occur. IV It is important to note that the chondrocytes of articular cartilage are not inert, and on electron microscopic examination they show an extensive endoplasmic reticulum and FIG. 2 Normal mature articular cazrtilage Golgi apparatus, indicating that they are proshowing arrangement of cells and matrix ducing protein and glycosaminoglycans. The in 4 zones. synthetic functions of chrondrocytes are:
Articular cartilage studies and osteoarthrosis (I) synthesis of protein for proteoglycans, collagen, and new cells; (2) synthesis and polymerization of glycosaminoglycans; and (3) sulphation of glycosaminoglycans. In osteoarthrosis the first observed histological abnormality is loss of matrix from the upper Zone II of the articular cartilage. This is followed by chondrocyte death, chondrocyte cluster formation, fibrillation of the cartilage, and erosion. Secondary changes -of thickening or ebumation, marginal osteophytes, and cysts-with alterations in blood flow which may be hyperaemia or venous stasis in different areas, occur in the underlying bone. The result of these changes is deformation of the surfaces of the joints and feeble repair response in the form of tufts of granulation tissue arising from the subchondral marrow. The mechanism of breakdown of articular cartilage is demonstrated schematically in Figure 4. Under the influence of compression-shearing stresses there is damage to cells in the upper Zone II of the cartilage. This results in the release of their catheptic enzymes, particularly
COMPRESSION SHEARING STRESSES
CELL DAMAGE mLY
MiNGLYCO$AIKOGLYCANS N.',;AND WATER LOSS
LOSS OF
CARTILAGE
RESILIENCY
FISSURES
CATIHEPSIN RELEASE
FURTHER CELL DAMAGE
L
LYSOMAL STABILISING AGENTS
RELEASE
Cartilage breakdown sequence in ostleoarthrosis, showing possible effect of lysosomal stabilizing agents. FIG. 4
89
cathepsin D, which are contained in the lysosomes of the cells. The cathepsin breaks the bond between the protein core and the glycosaminoglycans of the neighbouring matrix, resulting in loss of glycosaminoglycans. Hence there is water loss and loss of resiliency of the cartilage. As a result fissures occur. In the areas around the fissures further cells are subjected to excessive stress and are damaged. More enzymes are released from the lysosomes of these cells which digest more matrix; thus a vicious circle of breakdown of cartilage is produced and hence erosion of the articular cartilage occurs. The weight-bearing stress and movement of the joint surfaces cause a progressive breakdown of the articular cartilage. In response to the injury two processes occur in articular cartilage: (i) feeble chondrocyte proliferation and (2) increased synthesis of glycosaminoglycans. It is these two responses that I will consider first.
Repair of injuries limited to articular cartilage Mankin and Boyle4 have shown that when articular cartilage alone is injured by laceration of the surface there is a response in immature cartilage of proliferation of chondrocytes. This has been demonstrated by labelling the cartilage with tritiated thymidine, which is taken up in the S phase of mitosis. They found a mitotic index of i6o per I00 000 cells in newborn animals, but by the age of 6 months the mitosis in cartilage in response to injury had almost completely disappeared. Thus it was established that adult articular cartilage cells have almost no capacity for replication even after injury. However, Ash and Francis5 have demonstrated that when adult articular cartilage chondrocytes are isolated from their matrix and exposed to liver somatomedin in tissue culture the uptake of tritiated thymidine is
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George Bentley
greatly increased, which implies a greatly increased cell division rate. However, the healing of articular cartilage requires the formation of new matrix as well as cell division. In I965 Simmons and Chrisman' demonstrated that rabbits which were fed with sodium salicylate in therapeutic doses showed diminished breakdown of the articular cartilage of the knee after laceration. Also, by 8 weeks from the time of the laceration, in some animals healing of the cartilage was occurring which was not observed in controls. It was thought that the apparent healing in Simmons and Chrisman's experiment could have been related to the direction of the lacerations in the cartilage, since in different areas of joints the collagen fibre orientation in the superficial zone is such that it resists the major stresses. Thus it appeared that if the lacerations in cartilage were made in the direction of the predominant orientation of the collagen they would be more likely to heal than if they were made across the line of orientation. As a result a preliminary experiment was carried out on 24 mature rabbits. Twelve received lacerations in the femoral head articular cartilage in a radial direction (in line with the collagen fibres) and I 2 in a transverse direction. It was found that there was no healing of the cartilage in either group. A second experiment was performed on I4 animals, 7 of which received acetylsalicylic acid in their feed to produce a serum salicylate level of 1.09- .82 mmol/l (I 5-25 mg/ ioo ml). In this preliminary study examination of the cartilage sections showed that in those animals which had received a therapeutic dose of acetylsalicylic acid for 8 weeks healing of cuts had occurred (Fig. 5), whereas in the control animals it had not. These results suggest that acetylsalicylic acid has a healing effect on articular cartilage which
may be achieved either by diminishing breakdown of matrix due to lysosomal stabilization, thus allowing repair, or alternatively by stimulating matrix formation in some way. As a result of these investigations I have adopted the practice of treating all patients who have undergone arthrotomy of joints with possible cartilage damage-for example, those who have undergone meniscectomywith acetylsalicylic acid in therapeutic dosage for 8 weeks after operation. This is in an attempt to reduce the incidence of osteoarthrosis of the knee in patients who have undergone meniscectomy, which is known to be 25% over a period of 25 years7. It is im-
femoral head showing healing Of 3 lacerations after 8 weeks of oral. aspirin. (Toluidine blue, X 25.) mature rabbit
Articular cartilage studies and osteoarthrosis portant to realize that many other drugs have lysosomal-stabilizing properties-for example, glucocorticoids, chloroquine, inorganic gold salts, indomethacin, and phenylbutazone. Investigation of the effects of these drugs on articular cartilage is of great importance when considering the early prevention of cartilage breakdown and therefore of osteoarthrosis.
9I
papain to produce selective matrix breakdown appeared logical as a method of producing osteoarthrosis since the mechanism of its action is to break the bond of matrix proteoglycans between the protein core and the glycosaminoglycans of the side-chain in a manner comparable to that produced by cathepsin D (Fig. 6). This enzyme was employed, therefore, in an attempt to produce an experimental osteoarthrosis in mature
Experimental osteoarthrosis It is important for further study of the patho- animals8. Injections of 0.2 ml of 2% sterile papain genesis of osteoarthrosis and its response to various methods of treatment to observe solution activated by o.i ml of 0.03 M cystchanges at all stages in the development of eine solution were made into the hip joints the disease. Investigations into the effects of 40 mature Chinchilla rabbits on the ist, of different methods of treatment on joints 4th, and 7th days. The opposite hips received damaged by osteoarthrosis is restricted in the 3 injections of isotonic saline in the same living patient by the inaccessibility of the disx + eased tissues. Therefore there is a need for a model of osteoarthrosis which can be reproduced consistently in animals, which is recognizable without sacrifice of the animals, and which is produced in a manner similar to that occurring in human arthrosis. Table I shows the methods of inducing experimental osteoarthrosis which have been employed to date. All of these methods produce lesions which are not similar to osteoarthrosis or produce them by an effect which is not comparable to the mechanism of breakdown of articular cartilage in osteoarthrosis. The use of the proteolytic plant enzyme TABLE I Methods used for inducing experimental osteoarthrosis Lacerations, excision, percussion, and cautery Chemical (acids, alkalis) Foreign bodies (talc, cartilage fragments) Prolonged immobilization and compression Subluxations, dislocations, and relief of contact Hormones (growth, sex, steroids) Venous stasis Genetic Dietary fat
FIG. 6 Diagrammatic representation of action of papain and cathepsin D on proteoglycan molecule.
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volume as the right hip, as controls. The changes produced in the hip joints were studied at intervals from 6 h to 9 months by radiography, photography, and histological examination after staining with haematoxylin and eosin and with periodicacid-Schiff (PAS) counterstained with alcian blue to show the distribution of glycosaminoglycans.
Findings 6 h after one papain injection No radiographic changes were visible and the femoral head and acetabulum appeared normal to the naked eye. The articular cartilage over the superior surface of the femoral head and opposing part of the acetabulum was softer than on the control side when pressed with a blunt probe. Histologically, the cartilage of the upper surface of the femoral head showed slight fibrillation and indentation. There was loss of glycosaminoglycan staining of the matrix and also loss of nuclear staining of the chondrocytes, indicating their death, in Zones I and II (Fig. 7). The nuclei of some of the deeper chondrocytes were pyknotic. The acetabulum showed similar but less advanced changes. The changes in the articular cartilage at this stage resembled those observed in early
the central area-that is, Zones I-IIL-at the periphery the deep cells were more extensively affected than the superficial ones. The upper acetabular changes were more advanced than those at one day, with loss of matrix staining and chondrocyte death, occasional chondrocyte cluster formation, indentation of the surface articular cartilage, and the beginning of horizontal cleft formation. Findings 6 weeks after last papain The radiographs at this stage injection showed obvious narrowing of the joint space in the right hip, with slight sclerosis of the subchondral bone of the femur and acetabulum.
osteoarthrosis9"l0. Findings one week after last papain On naked-eye examination injection more widespread fibrillation of the articular surface of the upper femur was seen, with similar changes in the acetabulum. Histologically, loss of cartilage matrix staining was observed on the upper surface of the femoral head, with more extensive fibrillation of the articular cartilage than that seen after 6 h. Although the chondrocytes were dead throughout the thickness of the articular cartilage as far as the calcified layer in
FIG. 7 Section of articular cartilage of femoral head of mature rabbit 6 h after
intra-articular injection of 2% papain solution. (PAS and alcian blue, X 25.)
Articular cartilage studies and osteoarthrosis
93
was joint-space narrowing, sclerosis of subchondral bone of the femur and acetabulum, and subluxation of the femoral head. Small osteophytes were seen on the upper margin of the femoral head (Fig. 8). Macroscopic examination revealed extensive denuding of articular cartilage down to subchondral bone and similar though slightly less extensive damage to the acetabulum. Marked thickening of the joint capsule was present. The photomicrographs showed complete loss of articular cartilage down to subchondral bone in the weight-bearing areas. The subchondral bone was thickened and there was relative osteoporosis towards the margin of the femoral head; occasional subchondral cysts were seen (Fig. 9). Osteophytes were seen on the superior margin of the femoral head, with vessels and bone growing into the base of the articular cartilage. The acetabulum showed similar denuding of articular cartilage with underlying thickened subchondral bone. It is important to note that there was no sign of replacement of the surface of the femoral head or acetabulum by the remaining articular cartilage or from subchondral bone cells. At this stage the synovial membrane was Findings 9 months after last papa'n slightly inflamed and there were fragments injection The radiographic appear- of cartilage and bone engulfed and lying beances were advanced to the stage where there neath the surface. The femoral head showed flaking, fibrillation, and erosion of articular cartilage over the superior surface, as did the opposing acetabulum. Histologically, it was at this stage that the secondary response of the subchondral bone to the articular cartilage damage was first seen. The photomicrographs showed fibrillation and erosion of the articular cartilage on the upper surface of the femoral head extending into Zone III, but some of the deep chondrocytes remained alive. The most notable feature was the presence of new blood vessels invading the base of the articular cartilage at the periphery of the femoral head, with the formation of new bone. This process of endochondral ossification is an appearance very similar to that seen in human osteoarthrosis'0, and was the earliest manifestation of osteophyte formation in the experiment. The acetabulum showed similar but less advanced changes to the femoral head. The synovial membrane showed hyperplasia of the surface cells, and large fragments of articular cartilage were seen engulfed within it. The appearances were comparable to those seen in the synovium of osteoarthrotic joints'1.
FIG. 8 Radiograph of hips of mature rabbit 9months
after 3 injections of pap-
jJ
amn into right hip (see
~~~~text).
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George Bentley
FIG. 9 Section of femoral head 9 months after 3 in-
jections of papain (see text).
Table II shows the changes produced in papain arthrosis. It will be seen that the changes correspond to those seen in human osteoarthrosis in most features. The sequence of changes produced in this experiment demonstrate that advanced changes similar to those of human osteoarthrosis can develop in previously normal rabbit joints from primary changes induced in the articu-
lar cartilage matrix. This supports the view that osteoarthrosis developing in a previously normal joint is due to primary degeneration of articular cartilage rather than to changes in the subchondral bone.
Chondrocyte cluster formation in mature articular cartilage It has been noted that chondrocyte dcusters
TABLE II Changes produced in papain arthrosis compared with those in osteoarthrosis Papain arthrosis Osteoarthrosis Articular cartilage
Glycosaminoglycan loss Chondrocyte death and clumping Fibrillation Fissuring Erosion Subchondral bone Sclerosis Osteophytes Cysts Synovium Hyperplasia Cartilage and bone debris Increased vascularity Subsynovial fibrosis
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes
Yes Yes Occasional
Yes Yes
Yes Yes Yes Yes
Yes Yes Yes Yes
Yes
Articular cartilage studies and osteoarthrosis were seen in the articular cartilage within the first 24 h after the injection of papain. It has been argued on the one hand that they form as a result of multiplication of cells in response to damage of the cartilage and on the other that they represent passive aggregation of dead or dying cells secondary to loss of cartilage matrix. An experiment was carried out in which one injection of 2% sterile papain solution with 0.03 M cysteine as activator was made into the right hip joint of 4 mature New Zealand white rabbits. Isotonic saline was injected into the opposite hip as control. Rah bits were killed after 6 h and 24 h and the femoral heads and acetabula of both hips were incubated in a nutrient medium containing tritiated thymidine in a concentratinon of ioo mCi/l. Autoradiographs were prepared of serial sections of the femoral
95
heads and acetabula by the dipping technique of Joftes'2. It was found that chondrocyte clusters occurred in particular in the acetabula of the experimental hips after 6 and 24 h. Only occasional clusters were seen in the femoral heads. The counts from the serial sections were expressed as mitotic indices. The mitotic index of articular cartilage was increased in the papain-injected hip and to a slight extent in the opposite hip, presumably owing to a systemic effect of the papain. The number of mitoses fell after 24 h, suggesting that cartilage cell division was short-lived after the loss of matrix produced by one papain injection (Fig. io). It was noted that 5O% of the cells labelled in the autoradiographs were within chondrocyte clusters. This appeared to confirm that chondrocyte cluster formation was due to proliferation of articular cartilage cells. F
=
Ac.
Femur =
Acetabulum
* Experiment
Ac.R +
/
30-
Control
/
20*
Ac.R
10
/Ac
MITOTIC INDEX No. of Mitoses
12
/100,000 cells
1'2
6 TIME FROM INJECTION
-
AcL 1.
1824
HOURS
FIG. I0 Mitotic index of mature femoral head articular cartilage after one injection of papain into the hip.
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George Bentley
Repair capacity of arthrotic joints Next I would like to consider the response of joints which have lost articular cartilage, thus exposing the sibchondral bone, but which are not grossly distorted in their architecture so that clinically they are painful but still mobile. This is the stage at which many patients present for treatment, and the microscopic features correspond to those seen 8 weeks after 3 papain injections into a rab bit hip. The healing effect of intertrochanteric osteotomy of the arthrotic hip joint is well known but poorly understood. It has been observed that after femoral osteotomy bone cysts and sclerosis disappear from the femoral head on the radiographs, and the formation of a new joint space is observed in approximately 6o% of cases'3'14. It was decided to investigate these observed clinical phenomena experimentally. Two months after the induction of papain arthrosis in the hip joint I4 mature Chinchilla rabbits underwent high femoral osteotomy of the femur of a non-displacement type which was fixed with an intramedullary screw. This was to distinguish the biological effects of the osteotomy from any mechanical effects. Fourteen animals with papain arthrosis of the hips which had been induced at the same time were the controls. The animals in each group were divided into two subgroups, and after 3 months and 6 months respectively microangiography of the hip joints was performed by the intra-aortic injection of Colorpaque at a pressure of ioo mm Hg (I3.3 kPa). The animals were then killed and the femoral heads and acetabula examined histologically. The femoral osteotomies were united within 6 weeks of operation. Findings 3 months after femoral osteoIn 5 out of 7 animals which tomy had undergone femoral osteotomy micro-
FIG. II Microangiograms of femur: (a) 3 months after femoral osteotomy; (b) with papain arthrosis only.
angiography showed an increase in the number of arterioles in the femoral head and acetabulum compared with the controls (Fig. iI). No such increase was observed in the controls in comparison with normal hips. Thus the increase of arterioles was due to the osteotomy. Examination of the histological sections showed a consistent increase in the thickness of the subchondral bone plate of the femoral head in the osteotomy group, indicating increased formation of new bone. Findings 6 months after femoral osteoIn 6 out of 7 animals which tomy had undergone femoral osteotomy there was an increase in the number of arterioles in the femoral head and acetabulum despite the fact that the osteotomies were united by 6 weeks. Histologically, at this stage two types of surface repair of the denuded bone of the femoral head and acetabulum were seen. Those areas with small defects showed filling with fibrous tissue which was clearly emerging from the subchondral bone and undergoing cartilage metaplasia. Where there
Articular cartilage studies and osteoarthrosis
97
FIG. 12 Acetabulum from hip with papain arthrosis 6 months after femoral osteotomy. The surface is denuded of articular cartilage but 3 tufts of fibrocartilage are seen emerging from the subchondral marrow. (PAS and alcian blue, X io.)
Biological replacement of articular surfaces by transplantation of isolated chondrocytes Many joints affected by osteoarthrosis are damaged beyond the stage where induction of repair by the use of lysosomal-stabilizing drugs or juxta-articular osteotomy are of value. As a result many attempts have been made over the past 70 years to replace articular surfaces with grafts of articular cartilage It is thus possible to explain the pheno- and a thin layer of subchondral bone in both mena following femoral osteotomy in man as indicated in Figure I 3. The effect is augMECHANICAL EFFECT mented by the more favourable distribution BIOLOGICAL 4 EFFECT of stresses through the damaged area of the Increased vascularity I Redistribution of stresses joint after displacement osteotomy. 4 4 Increased New bone marrow The importance of these observations lies formation activity I in the possibility of inducing the changes of . Fibrocartilaqe I of hyperaemia, increased subchondral bone for- Clearinq formation cysts and mation, and joint-space repair by non- sclerosis 4 Joint healing to a lead could This means. operative l Pain relief of inmethods the of widening application ducing repair to other joints, where juxta- FIG. I3 Effect of femoral osteotomy on osteoarthrotic hip. articular osteotomy is not applicable.
were larger areas of denuding of the articular surface down to bone intense activity of the subchondral marrow cells was seen. Arising from these cells were tufts of fibrocartilage seen on the surface of the femoral heads and acetabula (Fig. I2). An extension of this fibrocartilage formation could have led to the formation of a continuous surface layer.
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humans and experimental animals'". There are two problems with such osteochondral allografts derived from, for example, cadavers. They are: (i) fixation of the grafts at the host site and (2) rejection of the graft by the host. A new approach to biological replacement of joints is therefore indicated. In I968 Chesterman and Smith"' grafted articular cartilage chondrocytes of adult rabbits, which had been isolated from their matrix by enzymatic digestion with papain and collagenese, into a defect cut in the upper articular surface of the humerus of adult rabbits. These allografts failed to survive. I decided to employ young cells taken from 6-week-old New Zealand white rabbits, since their capacity for cell division and matrix production is much greater than that of mature cells". The cells were isolated from the epiphysial growth plate of the metatarsals and also from the articular cartilage of the knee by digestion of the matrix with trypsin and collagenase and then, after gentle centrifugation at 5oo/min, transplanted into holes 3 mm wide and 3 mm deep drilled in the tibial articular surfaces of knee joints of adult rabbits of the same species. Transplantation into normal joints In the initial experiment the epiphysial cells and articular cartilage cells were each implanted into 8 mature rabbits. The controls were identical drill holes in the opposite knee which did not receive cells. The animals were killed after 8 weeks. The tibial condyles containing the grafts were incubated in nutrient medium containing IOO LCi of "5S-sulphate for 3 h at 370C and autoradiographs were then prepared. Histological sections 5 ,um thick were stained with alcoholic toluidine blue. Articular cartilage allografts Eight weeks after implantation of articular cartilage cells the defects appeared to be
FIG. 14 Allograft of immature articular cartilage 8 weeks after implantation. Graft is surrounded by inflammatory cells and fibrous tissue and is not incorporated. (Toluidine blue, X 7.5.)
filled with cartilage tissue in 2 of the 8 animals. Histologically, examination revealed that although there was cartilage present in 2 of the 8 defects it was not being incorporated, and intense inflammatory reaction was occurring, suggesting rejection of the grafted cells (Fig. I 4). Epiphysial cartilage allografts Eight weeks after implantation of epiphysial cartilage cells into the articular surfaces of rabbit tibae there was filling of the defects almost completely in 6 of the 8 animals. Histologically, the appearances were notable. There was filling of the defects with cartilage which was incorporated and which showed normal metachromasia of the matrix with toluidine blue staining (Fig. I5). In addition the autoradiographs demonstrated a normal distribution of 35S-sulphate, indicating that these cells were viable. The control drill holes showed only fibrous tissue loosely filling the
Articular cartilage studies and osteoarthrosis
99
physial and articular chondrocytes were implanted into drill holes in exactly the same way as in the normal joints and the animals were killed after 8 weeks. It was found that of the 8 joints which had received epiphysial cells complete filling of the defects had occurred in 3 and partial filling of the defect in a further 3. Of the 6 joints which had received articular cartilage cells partial cartilage filling had occurred in 2, but the remaining 4 showed only fibrous tissue in the defects FIG. I 5 Allograft of epiphysial cartilage 8 (Fig. I6). weeks after implantation. Cartilage fills the defect and is incorporated into host cartilage Conclusions It is concluded that the employment of epiand bone. (Toluidine blue, X 7.5.) physial cartilage chondrocytes as allografts for the filling of defects in articular surfaces may defects. There was no sign of rejection of have a clinical application, particularly if a the grafts. method of long-term storage of cells by deepproves possible. The reason for the survival of the epiphysial freezing that future biological reproposed It is cartilage cells, which has been confirmed in search into articular cartilage and osteofurther experiments, is not certain, but it may be because of their greater activity than articular cells for cell division and matrix production. Heyner'8 demonstrated that isolated chondrocytes can produce new matrix within io h of isolation from their original matrix and it is also known that the host antibody reaction requires approximately one week to become fully developed. It is proposed that epiphysial cartilage cells, because of their greater capacity for producing matrix compared with articular cells, can produce matrix rapidly enough to protect themselves from rejection by the host antigen-antibody reaction. Transplantation into joints with papain arthrosis The clinical application of this method of grafting is likely only if isolated chondrocytes can survive in an arthrotic joint. FIG. I 6 Allograft of epiphysial cartilage 8 Accordingly, in a small preliminary experi- weeks after implantation in knee with papain ment papain arthrosis was induced in the arthrosis. Healthy cartilage fills defect. (Toluknee joints of I4 mature rabbits. Isolated epi- idine blue, X 7.5.)
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arthrosis should be developed in five fields: i) The early diagnosis of osteoarthrosis in patients before radiological changes become apparent. 2) Prevention by lysosomal-stabilizing drugs of cartilage breakdown in patients whose joints are at risk from previous injury. 3) The production by growth hormone or somatomedin of repair in early cartilage damage. 4) Induction of fibrocartilage surface repair in damaged joints by osteotomy, surface drilling, or non-operative means such as hyperbasic oxygen therapy. 5) Replacement of grossly damaged surfaces by a technique of multiple drillings together with cartilage cell allografts. Although a cure is not yet available for ostcoarthrosis, pursuance of the avenues of research considered in this lecture should reduce the incidence of degeneration of articular cartilage and osteoarthrosis in those particularly at risk and reduce the necessity for extensive and sometimes difficult late reconstructive surgery in the future. I have pleasure in acknowledging the help given to me by Robert B Duthie, Nuffield Professor of Orthopaedic Surgery, University of Oxford, in whose department the above studies commenced anid finished. Professor A B Ferguson jr, of the University of Pittsburgh, generously provided a scholarship for one year. Technical help was provided by D W HIaynes, Mrs H Gorgescu, and Mrs I)ianne Deane. The illustrations were prepared by the photographic department of the Nuffield Orthopaedic Centre, and the manuscript was typed by Mrs Janet Lever.
References I
Heine, J (1926) Virchows Archiv fur pathologische Anatomie und Physiologie und fur klinische Medizin, 260, 52I.
I)anielsson, L G (I964) Acta orthopaedica Scandinavica, Suppl. 66. 3 Hunter J (1759) from John Hunter's manuscript (54) in the possession of the Royal Collegc of Surgeons of England. 4 Mankin, H J, and Boyle, C J (I967) Cartilage Degradation and Repair, p I85. Washington, D.C., National Academy of Sciences. 5 Ash, P, and Francis, M J (I974) Personal communication. 2
6 Simmons, D P, and Chrisman, 0 D (i 965) Arthritis and Rheumatism, 8, 960.
7 Jackson, J P (I967) Journal of Bone and Joint Surgery, 49B, 584. 8 Bentley, G (I97I) Journal of Bone and Joint Surgery, 53B, 324. 9 Collins, D H, and McElligott, T F (I960) Annals of the Rheumatic Diseases, 19, 3I8. IO
Harrison, M H M, Schajowicz, F, and Trueta, J (I953) Journal of Bone and Joint Surgery,
35B, 598. ii
Lloyd-Roberts, G C (1953) Journal of Bone and Joint Surgery, 35B, 627.
12
Joftes, D L (I959) Journal of Laboratory Inzvestigation, 8, I31.
13 Duthie, R B, and Howe, W W (I963) Clinical Orthopaedics and Related Research, 3I, 65.
14 Ferguson, A B jr (I964) Journal of Bone and Joint Surgery, 46A, II59.
I5 Bentley, G (I972) ChM Thesis, University of Sheffield. i6 Chesterman, P J, and Smith, A U (I968) Journal of Bone and Joint Surgery, 5oB, i84.
I7 Mankin, H J (I968) Bulletin of the Newv York Academy of Medicine, 44, 545. i8 Heyner, S (I969) Transplantation, 8, 666.
