Regenerating hyaline cartilage in articular defects of old chickens using implants of embryonal chick chondrocytes embedded in a new natural delivery substance

Calcif Tissue Int (1990) 46:246-253 Calcified Tissue International �9 1990 Springer-Verlag New York Inc.

Regenerating Hyaline Cartilage in Articular Defects of Old Chickens Using Implants of Embryonal Chick Chondrocytes Embedded in a New Natural Delivery Substance

Dror Robinson, 1 Nachum Halperin, 1 and Zvi Nevo 2

1Department of Orthopedics, Assaf Harofeh Medical Center; and 2Department of Chemical Pathology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel

S u m m a r y . Partial and full thickness defects were created mechanically in articular cartilage and subchondral bone of the tibiotarsal joint condyles of 3-year-old chickens. The wounds were then repaired using embryonal chick chondrocytes embedded in a new biocompatible, hyaluronic acid-based delivery substance. Controls were similarly operated on but received either no treatment or implants of the delivery substance only. Animals were killed from 1 week to 6 months postoperatively. Sections from the two groups were examined and compared macroscopically, histologically, and histochemical- ly. Results of 6-month follow-up showed that only the defects of the experimental chickens were completely filled with reparative hyaline cartilage tissue, with no signs of inflammation or immunologic rejection. Initially the entire defect cavity, whether partial thickness or full thickness up to the deep regions in the subchondral bone, was filled with cartilaginous reparative tissue. Relatively rapid matu- ration occurred under the tidemark; chondrocytes hypertrophied, were invaded with vascular elements and ossified. In the superficial areas, the reparative tissue remained cartilaginous and matured as typical hyaline cartilage tissue. These results in- dicate that aged chicken cartilage and its accompa- nying thin and spongy osteoporotic bone offer a fa- vorable host environment for embryonal cell implants.

K e y w o r d s : Articular cartilage regeneration - - Sub- chondral bone - - Implants - - Cultured chondrocytes - - Delivery system.

Send reprint requests to Z. Nevo, Department of Chemical Pa- thology, Sackler School of Medicine, Tel Aviv University, Ra- mat-Aviv, Tel-Aviv, Israel.

Sir James Paget first noted in 1853 that, "there are . . . no instances in which a lost portion of cartilage has been restored or a wounded portion repaired with new and well-formed permanent cartilage in the human subject." This view was later supported by Campbell [1] who examined numerous cartilaginous defects in several animal species as well as in humans and did not find normal cartilage within any. Ghadially [2], experimenting with rabbits, stated that, "Superficial injuries or partial thickness defects which do not violate subchondral bone do not heal, or rarely if ever heal, while deep injuries or full thickness defects which penetrate subchondral bone may heal by repair tissue arising from t h e

marrow spaces (and) . . . neither the size nor the site of the defect, the skeletal maturity or immatu- rity of the animal significantly affects the outcome of such injuries." The nature of such reparative tissue is controversial. Most authors agree that the final result is either fibrocartilage or hyaline cartilage of poor quality [1-4].

Attempts to repair articular cartilage defects using isolated chondrocytes have failed due to lack of adherence of the implanted cells to the lesion sites [5] or the formation of fibrocartilage or islands of cartilage embedded in fibrous tissue [5-7]. We re- cently reported the repair of mechanically created full thickness lesions (which transversed the subchondral bone plate) in the articular cartilage of young (4-month-old) roosters using a mixture of embryonal chondrocytes and a biological resorbable immobilization vehicle (BRIV) [8]. In the defects implanted with this mixture, quite typical hyaline cartilage formed. Animals with untreated defects or defects treated with BRIV alone showed no repair or repair with fibrous tissue.

In the present study, the previous fibrinogen- thrombin-based delivery system is replaced by a

D. Robinson et al.: Regenerating Hyaline Cartilage 247

n e w b i o c o m p a t i b l e - b i o d e g r a d a b l e , h y a l u r o n i c a c i d - b a s e d s u b s t a n c e . F u r t h e r m o r e , t h e c a r t i l a g e r e p a i r in sma l l d e f e c t s o f y o u n g a n i m a l s is e x t e n d e d b y t h e u s e o f a g e d (3 -yea r -o ld ) c h i c k e n s , as w e l l as t h e c r e a t i o n o f l a r g e r a n d i r r e g u l a r d e f e c t s , w h i c h

d i d n o t t r a n s v e r s e t h e s u b c h o n d r a l b o n e , m o r e

c l o s e l y a p p r o x i m a t e s t h e c l in i ca l s i t u a t i o n in os - t e o a r t h r o s i s . D e f e c t s t h a t d o n o t v i o l a t e t h e s u b - chondral bone plate do not have any capability for self-repair [2]. As most patients suffering from os- teoar throsis are old, the use of older animals seemed a more critical test of this technique. Such animals have been shown to have an even lower capability of self-repair of cartilage lesions than younger ones [9].

Materials and Methods

Implant Preparations

Epiphyseal chondrocytes of 11-day-old White Leghorn chick embryos were isolated and cultured in suspensions, as described previously [8]. Pellets of cells obtained from these cultures were mixed in a viscous preparation of 2%, high-molecular weight, hya luronic-ac id (BTG-BioTechnology General , Kir iat- Weizmann, Rehovot 76326, Israel) in phosphate-buffered saline (PBS). The cells were distributed evenly in the delivery substance at concentrations ranging from 5 • 10 6 to 10 X 10 6 celts/ml (Delivery substance-implant preparation, Patent #91080, 1989, Israel).

Material

Hyaluronic-acid (sodium hyaluronate) made by BTG is a high molecular weight (2-3 megadaltons) product, which is highly pu- rified (protein content <0.2 mg/g; pyrogens content <1.25 pg/g).

In vitro studies using chick embryonal chondrocytes showed that the delivery substance in PBS is not cytotoxic to the ceils and can support survival of cells in the preparation, outside an incubator at room temperature, for a period of up to 5 days. Ceils so treated for 5 days and then assayed incorporated S 35 into typical cartilaginous proteoglycans. When examined under a microscope, these cells appeared as typical embryonal chondrocytes and excluded trypan blue.

Operative Technique

Surgery was performed on 60 3-year-old Leghorn chickens under general anesthesia, which was induced by an intravenous dose of 60 mg/ml Nembutal (Ceva, Paris, France). The area of the right tibiotarsal joint was plucked, and the skin was prepared with a solution of povidone-iodine. The tibiotarsal joint was exposed through a lateral longitudinal incision and extended to provide exposure of the condylar articular cartilage. The patella was dis- located. In 30 of the chickens, a bone biopsy needle of 1.5 mm diameter was used to create a deep defect in the articular surface; this was then enlarged, using a gauge, to approximately 3-4

mm in diameter. Four to six such holes were created in the weight-bearing area of each operated joint. In the other 30 chickens, large and superficial defects were made using a scalpel.

The area occupied by the defect was calculated by photo- graphing the articular surface using a binocular microscope (Bausch and Lomb, StereoZoom-6-Photo-Microscope, Roches- ter, NY) and measuring the lesion's dimensions with a micrometric grid. A single deep defect occupied an average of 7.2% (range 6.1-8.0%) of the total articular surface or 12.8% (range 11.3-13.9%) of the weight-beating area of the tibia. The total area occupied by deep defects averaged 60% (range 51.3%--67.8%) of the weight-bearing area, or an average of 43% (32-48.1%) of the tibial articular surface. A superficial defect covered 26% (range 21.2-34%) of the total weight-bearing area of the tibia. Two defects were created in each joint. The total area occupied by the shallow defects averaged 63% (range 49.2%--69.2%) of the total weight-bearing area of the tibia.

The volume occupied by the defect was calculated by measuring the amount of glycerol needed to completely fill typical defects made using the same technique in an isolated joint specimen. The volume of an average deep defect was 40 _+ 20 ttl, whereas that of an average superficial one was 15 -+ 10 Ixl.

The animals were then divided into two groups of 40 experimental subjects (in 20 of them, deep defects had been made and in the rest, shallow ones) and 20 controls (in 10 of whom deep defects were created). In the experimental group, the wounds were filled with cultured embryonal chondrocytes embedded in the delivery substance. Defects created in the joint of animals in the control group were implanted with the delivery substance only or were not implanted at all. The contralateral tibiotarsal joint served as a control for histologlc comparison with normal chicken cartilage.

Postoperatively, the animals were housed in metal cages and kept at constant room temperature (23~ with a natural light cycle. Water and food were provided ad libitum. Animals were killed with an overdose of intravenous Nembutal at various time intervals, ranging from 1 week to 6 months after transplantation. Prior to dissection, the mobility of the operated tibiotarsal joint was checked and compared to the contralateral joint.

Histological and Histochemical Procedures

After the animals were killed the joint surface was photographed and immediately fixed in 4% formalin-PBS solution (pH 7.2) containing 0.5% cetylpyridinium chloride (CPC) for 48 hours and then in 4% formalin in PBS. After decalcification by repeated formic acid extraction, the specimens were dehydrated and embedded in paraffin. Five micrometer-thick sections were cut for histological and histochemical purposes. Sections were stained with Mayer's hematoxylin-eosin (H & E) Masson's trichrome (Masson's) stain for connective tissue constituents and Alcian blue stain (pH 1.0) for the detection of sulfated glycosaminogly- can (GAG). Sections cut through the defect were compared with sections cut through the same area in the contralateral (nonop- crated) joint. Histomorphometry was performed using a binocular microscope as well as an inverted microscope for larger mag- nifications (Olympus Microscope MIT-313, Olympus Optical Co. Ltd., Tokyo, Japan) fitted with a micrometric grid. Specimens were examined at x8, x40, • 100, x200, x400, and x 1,000 magnification. For every specimen, five nonconsecutive sections were assessed and their results averaged. In every section at least three nonovedapping fields were examined and their results averaged.

248 D. Robinson et al.: Regenerating Hyaline Cartilage

Cell density was calculated as the number of nuclei within a standard square area [10]. A mitotic ratio was calculated by dividing the number of mitoses seen in a standard area by the number of nuclei in the same area. Cartilage viability was assessed by calculating the number of normal-appearing nuclei by the total number of lacunae seen (which includes also empty lacunae as well as lacunae occupied by cells with pyknotic nuclei). The ratio of area occupied by matrix to that occupied by cells was calculated as well.

Results

General Clinical Observations

More than 95% of the chickens recovered well from anesthesia and started limping 1 hour to a few hours after the operation. The limp decreased gradually and at about 1 week postoperatively most chickens walked normally. At the time of sacrifice, no limited range of movement was observed in the operated joints. Wound infection developed in 2 chickens, which resulted in osteomyelitis.

Macroscopic Observations

The defects of the control group (untreated or delivery substance only) had sharp well-defined margins 1 week after operation. In some cases the defects were partially filled by a blood clot. In animals killed later than 1 month postoperatively, the defect either remained empty or was partially filled by a yellowish soft connective tissue clearly distinguishable from normal cartilage by consistency, color, and shine. This type of filling persisted throughout the 6-month observation period.

Seventy five percent of the defects of both types (deep and superficial) made in animals of the experimental group (chondrocytes in delivery substance) were filled with a whitish material (Fig. 1A and B). Of these, 40% were completely filled with this material, and the articular surface was perfectly restored. The remaining 35% were only partially filled, and the joint was not completely congruent. After I month, the successfully implanted defects were filled with a shiny material of bluish-white color and elastic consistency similar to that of the neighboring normal cartilage. Two months postoperatively, the defects were barely recognizable, and in some cases could not be identified with certainty. Their surface was even with that of the normal cartilage and they were distinguishable only by the dif- ferent light reflectivity. Success rate of the proce- dure was similar in both the large and shallow defects (Fig. 1B) and the small and deep ones (Fig. 1A).

Fig. 1. (A) Macroscopic photograph of operated distal tibial articular surface from experimental animal 6 months postoperatively. Four deep holes are visible (arrowheads) covering about 40% of the weight-bearing area. • (B) Macroscopic photograph of operated distal tibial articular surface from experimental animal 6 months postoperatively. Two large and shallow defects are visible covering about 50% of the weight-bearing area. x 8.

Microscopical Observations

When sectioned longitudinally, the area occupied by a single deep de fec t was 13.7% (range 10.2-15.6%) of the total area of the condyle; the superficial ones occupied about 19.6% (18.2- 21.3%). The depth of the deep defects averaged 4.2 mm (range 3.9--4.4 mm); the superficial defects averaged 0.9 mm (0.8-1.0 mm). Microscopically, the control defects were typically either empty or filled with a blood clot during the initial period (1-2 months after operation). Some of them eventually filled up with fibrous reparative tissue rich in vascular elements at the margins of the wounds (Fig. 2), with fine collagen fibrils scattered in the intercellular spaces of the spindle-shaped fibroblasts. A small percentage of the defects remained completely empty, with either well-circumscribed or roundish borders, even at 6 months postoperatively


Fig. 3. Low-power micrograph of a control defect 6 months after operation. The walls are still sharp, and an obvious crater is visible (asterisk). Some of the defect is filled with granulation tissue containing large blood vessels (arrows). H&E; •

Fig. 2. Low-power micrograph of a deep defect (asterisk) in a control animal 2 months after operation. Note the sharp walls of the defect (heavy arrows) and the formation of very little granulation tissue (thin arrows). Most of the defect remains empty (asterisk). H&E; •

(Fig. 3). In all cases , the control defects were clearly dist inguishable f rom surrounding normal cartilage and did not contain any areas of normal- appearing cartilage.

In the experimental group 1 week after surgery, the implanted defects were filled with an amorphous eosinophilic material, which contained scattered chondrocytes exhibiting high mitotic activity (Fig. 4). Two months after implantation, about 75% of the defects contained chondrocytes arranged in a lobular pat tern (Fig. 5), although the remaining 25% failed to grow cartilage and resembled the wounds of the control group. At the margins of the implanted regenerating cartilage, newly formed cartilage lay in direct apposit ion to the old neighboring articular cartilage (Fig. 6). In more than half of the defects, the cavity was filled completely with new cartilage, and the surface was continuous with that of the neighboring articular cartilage. At the deep

Fig. 4. One week after implantation, the defect is filled by delivery substance which appears as an amorphous eosinophilic material. In this material the chondrocytes are evenly distributed (arrows). Mitotic activity is evident (arrowhead). H&E; x 100.

regions, the onse t of endochondra l oss i f icat ion could be seen as early as 2 months postoperat ively (Fig. 7). The bony trabeculae adjacent to the im- p lan ted c h o n d r o c y t e s were normal and viable .


Fig. 5. Low-power micrograph of an experimental implanted defect 2 months postoperatively. The defect area is filled completely by newly formed cartilage. Note the lobular arrangement of the chondrocytes and the excellent integration of original and new cartilage surface (arrow). No trace of the delivery substance can be seen. H&E; •

There was no evidence of rejection around the implant. The most superficial chondrocytes were slen- der, had a clearer cytoplasm, and formed a horizontally oriented zone similar to normal articular cartilage. In shallow defects, the original subchondral bone plate was covered with cartilage. The joint surface appeared smooth and congruent (Fig. 8). Chondrocytes in the reparative tissue were embedded in matrix and stained intensively with Alcian blue (Fig. 9). In general, the implanted chondrocytes greatly resembled normal articular cartilage, but stained much stronger. No predominant fibrillar pattern could be demonstrated in the matrix using Masson's stain (Fig. 10), even at higher magnification.

In animals killed 6 months postoperatively, the endochondral ossification was much more ad- vanced. The grafts appeared well incorporated, and no signs of rejection were detected. The articular surface was smooth, and the most superficial layer

Fig. 6. Same histological slide as Figure 5 at a higher magnification. Note that the newly formed cartilage lies in direct apposi- tion to the original cartilage (arrow). H&E; x 100.

of chondrocytes was horizontally oriented, similar to the orientation of normal articular cartilage. Cell density in the normal chicken articular cartilage was approximately 50 (range 45-63) cells/mm 2. Immedi- ately after implantation, the cell density in the implant, consisting of delivery substance and embryonal cells, was 45 (37-58)cells/mm z. This increased to a peak of approximately 250 (180-300) cells/mm 2 I month after implantation. Later, as the cells matured and began to hypertrophy, their density declined to approximately 150 cells/mm z at 3 months; this cell density remained quite constant during the whole observation period. The number of mitoses of the implanted chondrocytes was highest immediately after implantation (-10%). This extreme rate rapidly declined to about 2.5% at 1 month after implantation. After 3 months, the rate of mitoses was only about 1% of all nuclei in the implant and this low rate seemed to remain constant during the observation period. In normal chicken articular cartilage, mitoses are exceedingly rare (rate <0.1%). The relative area occupied by matrix compared to that occupied by cells followed an opposite trend:


Fig. 7. At a high magnification, an area of endochondral ossification is seen. Hypertrophic chondrocytes (H) are surrounded by newly formed bone (B). H&E; •

Fig. 9. The cartilaginous reparative tissue, which formed in the defect, stains intensively with a specific dye for sulfated gly- cosaminoglycans (Alcian blue); •

Fig. 8. A superficial defect is repaired by grafted chondrocytes. Note that the subchondral bone plate (arrowheads) has not been transversed. The newly formed cartilage is closely integrated with the original cartilage (arrow) (two months after operation). H&E; x 100.

Fig. 10. Histological section of the defect and its surroundings, using Masson's trichrome stain which is a specific dye for the various extracellular matrix components. Note the smooth con- tour of the defect's surface. Masson's trichrome stain; x 100.

Immediately after implantation, the cells were embedded in the delivery substance with very little surrounding matrix. One month after implantation, cells occupied as much as 50% of the total area of the implant. This extreme cell density decreased to 30% after 3 months and about 20% after 6 months. Matrix occupied a much larger area of normal cartilage (90-95%). Eighty-four percent (80-100%) of lacunae contained viable-appearing nuclei in the implant, a rate similar to that observed in normal cartilage, and remained constant during the observation period.

Discussion

Articular cartilage, in animals as well as in man, has at best a highly limited self-repair ability, mainly

because of the lack of proliferation of the mature chondrocytes [8]. Autogenous repair, when and if it occurs, results in fibrocartilage, where fibroblasts are the principal proliferating cell type. Thus, a mixed tissue is formed containing both type I collagen (normally distributed in bone and ligaments) and type II collagen typical for articular cartilage [4]. Periosteum as well as perichondrium grafting to repair articular cartilage [11-15] also results in fibrocartilage in about 92% of cases [13, 14] without continuous passive motion of the joint; when passive movement of the joint is taken into account, the failure rate is still approximately 50% [13, 14]. In addition, the cartilage-containing tissue thus formed has little tendency to form palisades or align itself as normal cartilage does. After a long term, the new cartilage has a tendency to degenerate [11].


In a recent work, we considered embryonal chondrocytes advantageous for grafting procedures because of their documented superior proliferative ability [8]. The implanted chondrocytes are immu- nologically privileged due to their surrounding in- tact coat of extracellular matrix [16, 17]. The main problem, which to date has hindered all chondrocyte transplantation attempts, was the difficulty of immobilizing the cells in the implantation site. In- traarticular injection of a chondrocytes suspension did not result in the formation of cartilage, most probably because of lack of adherence [5, 7].

In previous experiments, our group demonstrated that the use of a mixture of embryonal chondrocytes and a gel-like biological glue composed of fibrinogen, thrombin, and an antiprotease substance (BRIV) enabled the repair of mechanically induced defects of articular cartilage [8]. In addition to immobilizing the cells, the glue inhibited unwanted cell invasion and granulation tissue formation during the critical first weeks after implantation. Sac- rifice of animals at short time intervals after the operation allowed us to show that the cells in the reparative tissue originated from the implant [8]. During the first few weeks after implantation, the grafted chondrocytes proliferated rapidly as a ho- mogeneous cell population. The BRIV was, how- ever, unsatisfactory in several respects. It was difficult to sterilize the highly concentrated fibrinogen solution, and many of the components of the glue were cytotoxic to chondrocytes in vitro (though leaving a residual cell population that proliferated in vivo after implantation) [8]. In addition, the consistency of the glue was such that it was difficult to obtain an even distribution of chondrocytes.

In the present work, BRIV was replaced by a simple one-component hyaluronate-based delivery substance. This material is easily sterilized, forms a hospitable microenvironment for chondrocytes, and is biodegradable. It can be produced at a range of consistencies and viscosities that allows an even distribution of the chondrocyte mixture. Another major advance of the present study was the exam- ination and repair of partial as well as full thickness defects in articular cartilage. Partial thickness defects, which do not transverse the subchondral bone, are notorious for their absolute lack of regenerative power because they are avascular [2]. Our success in resurfacing such defects proves that the newly formed chondrocytes originated from the implant and did not constitute an invasion of cells from the medullary bone or the bloodstream. In addition, shallow wounds are much harder to resur- face because their implants are exposed to strong shearing forces. We assume that our success was due to both the special properties of the delivery

substance and the young, actively dividing chondrocytes.

The utilization of implants containing embryonal chondrocytes to correct defects in articular cartilage is feasible in young animals as well as in aged ones. This view contrasts sharply with other works in which a complete lack of regenerative power in the cartilage of aged animals was found [9]. Further- more, it appears that the reparative tissue formed by the implants matures at a faster rate in old animals. The newly formed cartilage in the 3-year-old chickens grows faster and occupies larger spaces within the bone than in 4-month-old roosters, and the endochondral ossification in the deep regions occurs earlier and progresses more rapidly. These differences might be related to the osteoporotic bone of the older animals or, less likely, to the dif- ferent implant delivery substance used.

Our 75% success rate in resurfacing cartilage defects could conceivably be further improved by using continuous passive movement of the joint [14, 18]. Further research is essential in order to con- clude whether this method would be applicable in clinical practice.

In summary, the current work extends our previous observations and demonstrates the ability to induce cartilage regeneration in old animals, as well as in joints that lack a major or even most of their articular cartilage.

Acknowledgments: The authors thank Zoharia Evron of the Chemical Pathology Department, Pearl Alterman and Hana Abrahamer of the Histology Department for excellent and skill- ful technical assistance, and Asher Pinchasov for taking the pho- tomacrographs. This project was supported in part by a grant from the U.S.-Israel Binational Science Foundation.

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chondral defects in joint surfaces by neochondrogenesis with autogenous osteoperiosteal grafts stimulated by continuous passive motion. Clin Orthop 208:131-140

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Received November 18, 1988, and in revised form May 30, 1989.

Regenerating hyaline cartilage in articular defects of old chickens using implants of embryonal chick chondrocytes embedded in a new natural delivery substance

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