Basic to Clinical Cartilage Engineering: Past, Present, and Future Discussions 11

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Articular cartilage has limited capacity for self- repair and this inability may be the reason for the development of the end stage of cartilage loss, osteoarthritis (OA). OA is today a disability producer in many millions of people all over the world, and to find reliable methods of early repair of cartilage injuries and thereby prevent the development of OA seems of utmost importance. To learn more about how cartilage functions and how cartilage develops, studies on skeletal development during embryonic life could be helpful, especially as cartilage serves multiple functions in the developing embryo and those functions and events could tell us more about cartilage produced for postnatal life.

When a cartilage surface is injured, there are few chondrocytes that can start the repair partly because of the avascularity of cartilage and because of the low cell-to-matrix quota. But important knowledge that can be useful is that the embryonic cartilage development involves a mesenchymal high-density cell aggregation.^1–3 Subsequently, to imitate embryonic cartilage development, the goal for the orthopedic sur- geon is to try to deliver as many of the chondrogenic cells as possible into the cartilage lesion to achieve some sort of repair.

In Gothenburg, Sweden, the experience with biological articular resurfacing dates back to the early 1980s and the research has been focused on using the autologous articular chondrocytes^4–6 for local cartilage defect repair even though other chondrogenic cells such as aller- genic chondrocytes, auricular chondrocytes,

nasal chondrocytes, autologous rib perichondr- ial cells, periosteal cells, or mesenchymal bone marrow stromal cells could have been possible alternatives. However, the articular chondrocytes are responsible for the unique features of articular cartilage; therefore, it seems rational to use true committed chondrocytes to repair a cartilaginous defect.

Autologous Chondrocyte Transplantation

Cell Harvest

Biopsies of articular cartilage can be harvested by arthroscopy from a minor load-bearing area in the affected joint and at the same time 10 × 10 mL of autologous venous blood can be collected for preparation of serum additive to the culture medium.^7,8To get an idea of the amount of cartilage needed to be harvested, biopsies from 1000 patients were studied. The mean value of biopsy weight was 280 mg (4–1700 mg).

Furthermore, the mean value for cells/mg studied on 500 patients was 2600 cells/mg.⁹

Normally, the biopsies are taken as full thickness, through all layers down to the subchondral bone.^4,5However, recent studies by Dowthwaite et al.¹⁰tell us of the existence of a progenitor cell from the surface zone of articular cartilage. That population of cells exhibits high affinity for

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Basic to Clinical Cartilage Engineering: Past, Present, and Future Discussions

Mats Brittberg, Tommi Tallheden, and Anders H. Lindahl

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fibronectin, possesses a high colony-forming efficiency, expresses the cell fate selector gene Notch 1, and also exhibits phenotypic plasticity.

There seems to be a chondrocyte subpopulation with progenitor-like characteristics in the surface layer of cartilage indicating that it might be enough to harvest the superficial layers for chondrocyte isolation and expansion.¹⁰

In Vitro Cell Expansion

From such a piece of harvested cartilage, chondrocytes can be isolated by enzymatic digestion and the primary goal of in vitro chondrocyte expansion is to increase the cell number without loosening the redifferentiation ability of the cells. Individual chondrocytes are isolated by collagenase digestion overnight and cultured in Dulbecco’s modified Eagle medium/F12 with 10% autologous serum supplement. Primary cultures are performed in 25-cm²culture flasks and after 1 week the cells are subcultured by trypsin to 75-cm²culture flasks at a cell density of 8000 cells/cm²and the cells are cultured for another 1–2 weeks. During this period, the initial cells are expanded to 20–50 times the initial amount of cells.^4,5

The culture environment favors fast prolifer- ating cells and therefore it is possible that certain subpopulations could be concentrated.

Whether this is an explanation to why chondrocytes during in vitro culture lose their phenotype if the cells actually revert to a fetal stage, needs to be further elucidated.

Condensation

During the initial 12-week period of embryonic life, the formation of the skeleton begins. The beginning process involves a mesenchymal cell aggregation, which is the fundamental feature of cartilage differentiation in the developing limb with the formation of the prechondrogenic cell condensation, a blastema.¹In in vitro studies, it has been shown that mesenchymal cell aggre- gates must achieve a threshold size before chondrogenesis can proceed.²Subsequently, to start chondrogenesis in order to repair a cartilaginous tissue, a large number of chondrogenic cells for implantation in high densities are needed.

The dedifferentiated chondrocytes have a similarity to primitive mesenchymal cells and an

implantation of a high density of those in vitro- expanded primitive immature chondrocytes could imitate the prechondrogenic cell condensation and cartilage formation.⁶ The repair process initiated by implantation of the cells into the defect initiates cell condensation and cartilage formation similar to the mesenchymal condensation in limb formation discussed above. To evaluate that similarity, special primers for limited key genes expressed during limb and cartilage development have been studied by Lindahl et al.⁸: collagen type IIA and IIB, osteocalcin, SRY-related HMG box gene (Sox9), cartilage-derived morphogenetic protein 1, wingless type MMTV integration site family member 14 (Wnr-14), and core-binding factor alpha 1. All the described genes were expressed by the cultured cells and especially Sox9 and Wnt-14 expression were of interest because of their importance in embryonal cartilage cell condensation and joint development.⁸

Redifferentiation

Doubts have been expressed that the dedifferentiated cells would not revert enough to contribute to a repair after implantation. However, several researchers have shown that dedifferentiated chondrocytes are able to reexpress phenotypic markers of the articular chondrocyte.^11–13 The tests include three-dimensional cultures in gels of agarose, collagen, and alginate. The redifferentiation can also be obtained after treatment of chondrocytes in monolayer by dihydrocytochalasin B or staurosporine.^11,12 Tallheden et al.¹³ studied monolayer culture- expanded chondrocytes that redifferentiated in a pellet model as seen by an increase in collagen type II immunoreactivity between days 7 and 14.

The late phase consisted of a strong down-regulation of extracellular signal-regulated protein kinase (ERK-1) and an up-regulation of p38 kinase and Sox9, suggesting that the late phase mimicked parts of the signaling processes involved in the early chondrogenesis in limb bud cells. Other genes, which indicated a transi- tion from proliferation to tissue formation, were the down-regulated cell cycle genes GSPT1 and the up-regulated growth-arrest-specific protein (gas)¹³[Figure 11.1 (see color section)].

Although the mechanisms involved in restoration of the differentiated phenotype have not been elucidated yet, it has been shown that

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the synthesis of specific proteoglycans and type II collagen can be related to a modification of actin architecture.

Cell Implantation

The culture-expanded cells normally arrive to the operating room in syringes at a density of 30 million cells/mL.^7,8The damaged joint is opened and the cartilage injury is debrided to healthy cartilage. A flap of periosteum or a collagen membrane is sutured over the defect and finally the cells are implanted into the defect with a treatment dose of 1 × 10⁶cells/cm²of defect area [Figure 11.2 (see color section)].

The ability of the culture-expanded chondrocytes to adhere to the debrided surface has been questioned but several studies in vivo and in vitro have shown that the chondrocytes are able to adhere and redifferentiate within the joint. Chen et al.¹⁴ used radiolabeled chondrocytes for quantification and showed that the efficiency of transplantation onto a cartilage substrate was 93% ± 4% for seeding densities of as much as 650,000 cells per cm²after a seeding duration of 1 hour. During the 16 hours after seeding onto a cartilage substrate, the transplanted cells synthesized sulfated proteoglycan in direct proportion to the number of cells seeded. Most (83%) of the newly synthesized proteoglycan was released into the medium rather than retained within the layer of transplanted cells and the recipient cartilage substrate, thus suggesting that at least early on, an overlying membrane is important.¹⁴

These results implicate that, at least in experiments with chondrocytes directly seeded to cartilage explants, a linear relationship between biosynthetic activity and the number of seeded chondrocytes could be seen, which means that the number of seeded cells seems important.¹⁴ However, the number of cells clinically needed for the implantation either as a suspension or in a scaffold has not been studied enough.

LeBaron and Athanasiou¹⁵ noted that polylac- tide-polyglycolide scaffolds seeded with a density of less than 10 million cells per milliliter, resulted in very little cartilaginous material.

They stated that seeding at high cell densities seemed advisable.¹⁵Puelacher et al.¹⁶presented a study that revealed that seeding scaffolds at a density ranging from 20 to 100 million cells per milliliter resulted in formation of clinically

appropriate cartilage when implanted into nude mice subcutaneously.

In another study,¹⁷ histologic analysis indicated that chondrocytes grown on devitalized cartilage discs produced new matrix that bonded and integrated individual cartilage ele- ments with mechanically functional tissue.

Biomechanical testing demonstrated a time- dependent increase in tensile strength, failure strain, failure energy, and tensile modulus to values 5%–30% of normal articular cartilage by 8 months in vivo. The values recorded at 4 months were not statistically different from those collected at the latest time point, indicating that the limits of the biomechanical property values were reached after 4 months from implantation.

Sohn et al.¹⁸used an ex vivo bovine model to study the implanted chondrocytes’ behavior and orientation relative to gravity of a repaired full- thickness articular cartilage defect and found that the gravity affected the initial distribution of transplanted chondrocytes, prelabeled with 3H-thymidine. Those results indicate that injected chondrocytes localize under the influence of gravity within the initial few hours after injection. Therefore, the defect orientation during this time can be an important factor in the uniformity of cell distribution in the autologous chondrocyte implantation procedure and may be an important determinant of the ultimate clinical outcome.

To monitor the persistence and the phenotype of injected chondrocytes in repair tissue, Dell’Accio et al.¹⁹ used a fluorescence labeling protocol for articular chondrocytes, which allowed cell tracking in vivo using the fluorescence dye PKH26. Their data indicate that the implanted cells can persist for at least 14 weeks in the defects, can participate in integration with the surrounding tissues, and become a structural part of the repair tissue rich in collagen type II and sulfated proteoglycans.

Cell-to-Cell Contact and Integration to the Surrounding Tissue

The initial attachment of transplanted chondrocytes to the surface of a cartilage defect is crucial for the success of chondrocyte transplantation.

The attachment is important regarding the start of chondrogenesis, induction of the differentiation

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program, and integration of neocartilage produced into surrounding native cartilage.

Wang and Kandel²⁰used freshly isolated primary or passaged chondrocytes that were seeded on the top of bone plugs having a surface composed of mid-deep zone hyaline cartilage or calcified cartilage or onto bone only. Both primary and passaged chondrocytes attached effi- ciently to all three surfaces (>88% of seeded cells). As a sign of start of a chondrogenesis, there was expression of type II collagen and aggrecan core protein mRNA by 2 hours postimplantation.

Kurtis and coworkers²¹studied the time influence on cell attachment. After culture in monolayer, adhesion of chondrocytes to cartilage increased with time, from 6%–16% at 10 min- utes to a maximum of 59%–82% at 80–320 min- utes. They also found that β1, α5 β1, and αvβ5 integrins seem to be involved in human chondrocyte initial adhesion and retention to cartilage. Enzymatic treatment of a cartilage surface can further enhance the chondrocyte adhesion to surrounding native cartilage.

Tacchetti et al.²²have shown that with chick embryo-derived undifferentiated cells, a reduced rate of cell clustering and cell-to-cell contact parallels a reduction of cell recruitment into the cartilage differentiation program. They suggested that there was a cascade of events important in the early stages of chondrocyte differentiation consisting of the acquisition of the ability to establish cell-to-cell contacts, the formation of a permissive environment capable of activating the differentiation program, and finally the expression of differentiation markers.

Gene Therapy

To alter or improve the cells before implantation or by directly changing the function of the chondrocytes in situ, gene therapy is a possible option. Different cDNAs have been cloned, which may be used to stimulate biological processes that could improve cartilage healing by (1) inducing mitosis and the synthesis and deposition of cartilage extracellular matrix components by chondrocytes, (2) induction of chondrogenesis by mesenchymal progenitor cells, or (3) inhibiting cellular responses to inflamma- tory stimuli. It is possible that the chondrogenic cells transfer genes encoded for cartilage repair promoting factors directly to the lesion site. The

transferred genes must enter the cell’s nucleus where it can be transcribed followed by a trans- portation of the generated mRNA out from the nucleus to serve as matrix for protein production in the ribosomes.

Viral gene vectors are regarded as more effi- cient than nonviral vectors such as liposomes.

Milbrandt et al.²³ explored the feasibility of using transduced cells for gene therapy to induce healing of osteochondral defects. Both a mouse mesenchymal cell line and mixed rabbit adherent stromal cells were transduced with either liposomal transfection or retroviral transduction using a traceable gene. Transduction efficiency was more than 95% with the retroviral construct and expression was maintained for more than 6 months of passage. The liposomal transfection led to a transient expression with an efficiency of 50%. The expression of osteochondral genes was diminished but preserved after transduction in vitro. Transduced rabbit cells were transplanted into osteochondral defects in rabbit femoral condyles. Cells transplanted in vivo could be detected for 4 weeks in the repair tissue. Milbrandt et al.²³ demonstrated that mesenchymal cells from bone marrow, stably transduced with a traceable gene product, retain the bone and cartilage phenotype and can be followed in vivo after transplantation into cartilage defects. Similar animal experiments clearly demonstrate the utility of tissue engineering strategies in which gene therapy is used to locally influence the repair environment.

One drawback with gene therapy is the relatively short half-lives in vivo of the gene expression, which often require high doses and repeated injection, and still much research remains until a safe clinical procedure can be used.

Growth Factors

Relatively little is known about the intracellular signaling pathways involved with growth factors and cartilage repair processes but one important group of growth factors that have raised special interest are the peptide growth factors. Of these factors, there are three growth factor classes that are especially interesting, the fibroblast growth factor family, the insulin-like growth factors including insulin, and transforming growth factor β (TGFβ) and related molecules.^24,25All these factors have been implicated in three aspects of

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cartilage growth and metabolism: The induction of mesoderm and differentiation of a cartilaginous skeleton in the early embryo, the growth and differentiation of chondrocytes within the epiphyseal growth plates leading to endochondral calcification, and the processes of articular cartilage damage and repair.²⁴Repair of defects in tissues normally requires regeneration of injured cells via control of the cell cycle, differentiation, and cell death (apoptosis). The peptide growth factors usually work through paracrine effects and are able to initiate cell replication through cell surface receptors and thereby initiate cascades that amplify the cell’s response.

Because of the poor in situ capacity of cartilage repair, it would be ideal if growth factors could be loaded into a device designed to stimulate the cells already present in the joint to repair the injury. Potential candidates are TGFβ and related bone morphometric proteins, which can induce the differentiation of cartilage from primitive mesenchyme, and together with basic fibroblast growth factor and insulin-like growth factors promote cartilage growth.^26–28Of interest also are other members of the TGFβ superfamily that are thought to have key roles in chondrocyte growth and differentiation. Notably, bone morphogenetic protein-2 (BMP-2) may be a useful cytokine to improve healing of cartilaginous defects.^28–30 The problem, however, is that the control of chondrocytic differentiation of primitive stem cells is affected by interplay and expression of several factors such as Brachyury, BMP-4 (BMP-2B), TGFβ3. Important for the phenotypic expression is also the Smads 1, 4, and 5.³¹ The situation is therefore rather com- plex and, instead of a single growth factor at one occasion, several factors in a cascade are needed.

Animal Experiments

In 1984 and 1989 Peterson et al.³²and Grande et al.,³³ respectively, presented their results in rabbit models with autologous chondrocytes grown in vitro on the healing rate of chondral defects not penetrating the subchondral bone plate. In defects that had received transplants 8 weeks’ postimplantation, a significant amount of cartilage was reconstituted (82%) compared with engrafted controls (18%). Autoradiography on reconstituted cartilage showed that there was

labeled cells incorporated into the repair matrix.³³ Goldberg and Caplan³ compared implantation of mature chondrocytes, so-called committed, with mesenchymal stem cells for the repair of full-thickness defects in the femoral condyles of adult rabbits. Both cell types repaired the defect with hyaline-like neocartilage. However, the mesenchymal stem cell repair had a more hyaline-like morphology and cartilage zonal characteristics than the repair from the committed chondrocytes. Mesenchymal cells as chondrogenic progenitors have also been studied by Nevo et al.,³⁴who found prob- lems such as a delayed pace of endochondral ossification in the deep zones of the subchondral region of the study defects, or ossification above the tide mark, within the superficial cartilaginous articular regions. They found that autogenetic, chondrocytic-enriched bone marrow-derived mesenchymal cells were superior to other cell sources for articular cartilage regeneration.

Brittberg et al.⁵ compared periosteal grafts with and without chondrocytes in rabbit patellar chondral defects. One year after implantation, periosteum and chondrocytes demonstrated an average repair area of 87% compared with the 30% repair area observed in the periosteum- alone graft. A histologic grading system also demonstrated a statistically significant differ- ence between the two graft types, with the tissue produced by chondrocytes in combination with a periosteal flap resulting in much higher histologic scoring compared with defects treated with only a periosteal flap.

Rahfoth et al.³⁵ transplanted allograft chondrocytes embedded in agarose gel into rabbit articular cartilage defects and followed up the repair for 6–18 months. At 18 months, in 47% of the grafts, a morphologically stable hyaline-like cartilage was noted to develop, and extent of recovery was never observed in controls.

Using a dog model to study autologous chondrocyte repair on chondral repair, Breinan et al.³⁶found that, at 12–18 months postsurgery, there were no differences in the repair with autologous chondrocytes and periosteum versus periosteum alone, but a substantial number of animals had a break through to the subchondral bone.

Recently, using the previously established canine model for repair of articular cartilage defects, Lee et al.³⁷evaluated the 15-week healing of chondral defects (e.g., to the tidemark)

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implanted with an autologous articular chondrocyte-seeded type II collagen scaffold that had been cultured in vitro for 4 weeks before implantation. The amount and composition of the reparative tissue were compared with results from the Breinan study using the same animal model in which the following groups were ana- lyzed: Defects implanted with autologous chondrocyte-seeded collagen scaffolds that had been cultured in vitro for approximately 12 hours before implantation, defects implanted with autologous chondrocytes alone, and untreated defects. Chondrocytes, isolated from articular cartilage harvested from the left knee joint of six adult canines, were expanded in number in a monolayer for 3 weeks, seeded into porous type II collagen scaffolds, cultured for another 4 weeks in vitro, and implanted into chondral defects in the trochlear groove of the right knee joints. The percentages of specific tissue types filling the defects were evaluated histomorpho- metrically and certain mechanical properties of the repair tissue were determined. The reparative tissue filled 88% ± 6% of the cross-sectional area of the original defect, with hyaline cartilage accounting for 42% ± 10% (range 7%–67%) of defect area. These values were greater than those reported previously for untreated defects and defects implanted with a type II collagen scaffold seeded with autologous chondrocytes within 12 hours before implantation. Most interesting was the decreased amount of fibrous tissue filling the defects in the current study, 5% ± 5% as compared with previous treatments.

Despite this improvement, indentation testing of the repair tissue formed revealed that the compressive stiffness of the repair tissue was well below (20-fold lower stiffness) that of native articular cartilage.

Still, long follow-ups with a reliable and maybe larger animal are lacking. However, it appears that the above different animal models with allogenous or autologous chondrocytes used in the treatment of cartilage defects show benefit over cartilage defects treated without cells or not treated at all, at least in reports of little more than 1 year’s follow-up.

The Clinical Research

Clinically, the first patients with articular cartilage defects were treated by implanting autologous cells expanded in vitro in combination with a biomechanical membrane – the periosteum in

1987.⁴This, the first generation of chondrocyte transplantation, was initially termed autologous chondrocyte transplantation (ACT). Because the periosteum itself has chondrogenic potential,³⁸a dual chondrogenic response when using the chondrocytes in combination with the periosteum may be expected. Today, the technique is called either ACT or ACI (autologous chondrocyte implantation). In Sweden, autologous chondrocyte transplantation in combination with a periosteal graft has been used in approximately 1200 patients since October 1987 and, worldwide now, variants of ACT/ACI have been tried in approximately 12,000 patients. The use of ACT is always considered alongside other techniques.

In patients with small, acute defects, ACT is mostly used after failure of other techniques after 6 months. In large, acute defects, ACT and osteochondral grafting (up to 4 cm²) may be used immediately because such large defects are difficult to resurface with bone marrow stimula- tion techniques.

In a clinical evaluation of 244 patients with 2–10 years’ follow-up,⁹subjective and objective improvements were seen in high numbers of patients with femoral condyle lesions and osteochondritis dissecans. There was a high percentage of clinical good to excellent results (84%–90%) in patients with different types of single femoral condyle lesions whereas other types of lesions had a lower degree of success (mean 74%). Biopsies of repair tissue showed mostly a mixed repair tissue with a fibrous top layer and more hyaline-like middle and bottom layer [Figure 11.3 (see color section)].

To study the long-term durability of ACT- treated patients, 61 patients that had passed 2 years postsurgery were followed for at least 5 years up to 11 years postsurgery (mean 7.4 years). After 2 years, 50 of 61 patients were graded good–excellent.

At the 5- to 11-year follow-up, 51 of the 61 were graded good–excellent. The total failure rate was 16% (10/61) at mean 7.4 years. All ACT failures occurred in the first 2 years, so patients showing good to excellent improvement at 2 years had a high percentage of good results at long-term follow-up.

Most reports on the use of autologous chondrocyte transplantation from other centers show similar figures with a high degree of success in regard to the total number of improved patients but the criticism has been that ACT needs to be evaluated versus other cartilage repair techniques in randomized trials.³⁹ A very compre-

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hensive analysis of the ACT technology was recently presented.⁴⁰The authors stated that, on the basis of literature, no definite conclusions can be drawn about the clinical effectiveness of ACT, which should be regarded as an experimental procedure. However, on these grounds, almost all other techniques used for treating dis- abling cartilage defects, could be regarded as experimental. The cost of ACT is substantial in comparison to the other techniques, and surgeons have reported similar good results with those techniques as have been reported with ACT. Most of those other cartilage repair techniques have still quite short follow-ups (2–4 years) and the authors speculate that it is possible that, by extending the time horizon and assuming that better outcomes are sustained with ACT, but not with other therapies, financial and human costs might in the long run be less with ACT. Such speculations, according to the authors, are not justified until data from randomized studies become available. It is easy to agree with that statement, but you may empha- size the importance of long-term follow-ups in such studies with the definitions of a short-term study being a minimum of 2 years, mid-term 5 years, and a long-term study at least 10 years.

To date, three such clinical randomized trials have been reported on. Those studies as described below could be seen as short-term studies. Knutsen et al.⁴¹studied 80 patients who needed local cartilage repair because of symptomatic lesions on the femoral condyles measur- ing 2–10 cm². The patients came from four hospitals and were randomized into ACT or microfracture treatment and followed at 12 and 24 months. At 2 years, both groups had significant clinical improvement. According to the SF-36 physical component score at 2 years post- operatively, the improvement in the microfracture group was significantly better than that in the autologous chondrocyte implantation group. Both methods had acceptable short-term clinical results. There was no significant differ- ence in macroscopic or histologic results between the two treatment groups.

Horas et al.⁴²performed a prospective clinical study to investigate the 2-year outcomes in 40 patients with an articular cartilage lesion of the femoral condyle who had been randomly treated with either transplantation of an autologous osteochondral cylinder or implantation of autologous chondrocytes. Both treatments resulted in a decrease in symptoms. However, the improvement provided by the autologous chondrocyte

implantation lagged behind that provided by the osteochondral cylinder transplantation.

Histologically, the defects treated with autologous chondrocyte implantation were primarily filled with fibrocartilage, whereas the osteochondral cylinder transplants retained their hyaline character, although there was a persist- ent interface between the transplant and the surrounding original cartilage. The authors mentioned the limitations of their study because it included a small number of patients, the relatively short (2-year) follow-up, and the absence of a control group.

Bentley and associates⁴³studied 100 patients with a mean age of 31.3 years (16–49) with symptomatic chondral and osteochondral lesions of the knee, which were suitable for cartilage repair, and were randomized to undergo either ACT or mosaicplasty: 58 patients had ACI and 42 mosaicplasty. Most lesions were post- traumatic and the mean size of the defect was 4.66 cm². The mean duration of symptoms was 7.2 years and the mean number of previous operations, excluding arthroscopy, was 1.5.

The mean follow-up was 19 months (12–26).

Functional assessment using the modified Cincinnati and Stanmore scores and objective clinical assessment showed that 88% had excellent or good results after ACT compared with 69% after mosaicplasty. Arthroscopy at 1 year demonstrated excellent or good repairs in 82%

after ACI and in 34% after mosaicplasty. All five patellar mosaicplasties failed. This prospective, randomized, clinical trial showed significant superiority of ACT over mosaicplasty for the repair of articular defects in the knee.

The ankle joint, shoulder, elbow, hip, and wrist^44–48are other locations that have been tried in smaller numbers of patients. With the development of arthroscopic techniques,^49,50the use of ACI will be increased also in those smaller joint compartments. Specially, the use of resorbable membranes instead of the periosteum will be important for those joints by which one may avoid the risk of hypertrophy. The periosteal hypertrophy may become a problem in the smaller joints.

The first generation of ACT is a combination treatment with two chondrogenic factors involved: The implanted suspension of chondrocytes and the cambium cells from the periosteum. The disadvantages include the potential leakage of cells from defects, dedifferentiation of cellular phenotype as the cells have been grown in monolayer just before implantation, and

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uneven distribution of cells as well as substantial risk of periosteal hypertrophy. Resorbable membranes, such as Chondro-Gide (a type I/type III collagen membrane, Geistlich, Pharma AG, CH- 6110 Wolhausen, Switzerland), are a possible alternative to the periosteum^51,52and the clinical results with that membrane seem promising.

Similar good short-term results have also been presented with the hyaluronan-based biodegrad- able polymer scaffold HYAFF-11(Fidia Advanced Biopolymers, Abano Terme, Italy).^53,54

The Future

We will see more and more different cell-contain- ing resorbable scaffolds to be used for arthroscopic implantation; some scaffolds with cells cultured for several weeks(mature grafts) whereas others just seeded with cells 1–2 days before sur- gery(immature grafts) or even just at implantation time. However, the repair is also dependent on a fast integration of the implant and immature constructs have poorer mechanical properties but integrate better than either more mature constructs or cartilage explants. Integration of immature constructs involves cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage, which involves only the secretion of extracellular matrix components.

Trypsin and chondroitinase ABC treatments of the adjacent cartilage further enhance the integration of immature constructs.⁵⁵

The ACT technology needs to be further improved and there is a search for new biomaterials, which are aimed to secure the cells in the defect area and permit their proliferation and differentiation. The maintenance of the original phenotype by isolated chondrocytes grown in vitro is an important requisite for their use and handling in a future transarthroscopic technique in articular cartilage resurfacing.

Conclusion

An analysis of the literature provides no evidence so far for regular regeneration of hyaline cartilage in animal experiments and still today’s treatments for cartilage resurfacing are less than satisfactory, and rarely restore full function or

return the tissue to its native normal state. The rapidly growing field of tissue engineering holds great promise for the generation of functional cartilage tissue substitutes. Cell biologists, engi- neers, and surgeons work closely together with combined knowledge of using biocompatible, biomimetic, biomechanical suitable scaffolds seeded with chondrogenic cells and loaded with bioactive molecules that promote time-relapsed cellular differentiation and/or maturation.

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