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Osteoarthritis and Cartilage 23 (2015) 334e350

Review An educational review of cartilage repair: precepts & practice e myths & misconceptions e progress & prospects

* E.B. Hunziker , K. Lippuner a, M.J.B. Keel b, N. Shintani c

Departments of Osteoporosis, Orthopaedic Surgery and Clinical Research, Inselspital, University of Bern, Bern, Switzerland

article info summary

Article history: Objective: The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty Received 10 July 2014 clinical problem. Although research activity in this area has been indefatigably sustained, no significant Accepted 12 December 2014 progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before Keywords: we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair Articular redivivus! Cartilage Design: Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational Repair Tissue engineering accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review Osteoarthritis addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, Review focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. Results and Conclusions: Tissue engineering aims to reconstitute a tissue both structurally and func- tionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be sur- mounted for ultimate success are discussed. © 2014 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

Introduction Articular cartilage is a highly specialized tissue. Lesions therein seldom heal, or heal only partially under certain biological condi- The bony ends of mammalian synovial joints are lined with a tions. They are frequently associated with symptoms such as joint layer of articular cartilage (Fig. 1), which permits congruency be- pain, joint-locking phenomena and reduced or disturbed joint tween the two opposing skeletal elements, facilitates the transfer of function. Moreover, the progression of such lesions is generally e forces between these and their practically frictionless movement, believed to forebode the onset of osteoarthritis (OA)2 4. and acts as an absorber of weight during sustained static loading1. In Western countries, OA is one of the most common diseases suffered by elderly persons. It is a disabling joint disorder of multifactorial aetiology, and, consequently, of usually unknown * Address correspondence and reprint requests to: E.B. Hunziker, Center of origin in individual cases (primary OA). However, it can be Regenerative Medicine for Skeletal Tissues, Departments of Osteoporosis, Ortho- precipitated also by physical injury [e.g., by sports or occupational paedic Surgery and Clinical Research, Inselspital, University of Bern, Murtenstrasse accidents (secondary OA)]. The incidence of OA is positively 35, P.O. Box 54, 3010 Bern, Switzerland. Tel: 41-31-632-86-85; Fax: 41-31-632- 49-55. correlated not only with age but also with obesity, and due to the E-mail addresses: [email protected] (E.B. Hunziker), kurt.lippuner@ upward trends in both life-expectancy and youth overweight, a e insel.ch (K. Lippuner), [email protected] (M.J.B. Keel), nahoko.shintani@dkf. steady increase in the prevalence of the disease is expected5 7. unibe.ch (N. Shintani). Although joint pain is a common concomitant of OA, this symptom a Tel: 41-31-632-31-28; Fax: 41-31-632-95-96. b Tel: 41-31-632-22-13; Fax: 41-31-632-18-80. is not correlated with the size of the articular-cartilage lesion, and 8e10 c Tel: 41-31-632-09-37; Fax: 41-31-632-49-99. the mechanism underlying its generation is largely unknown .

http://dx.doi.org/10.1016/j.joca.2014.12.011 1063-4584/© 2014 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 335

Lesioning of the articular-cartilage layer

As aforementioned, structural lesioning of an articular-cartilage layer can be induced either traumatically or during the course of a disease-based process. Once the process of lesioning has begun, it cannot be arrested, but progresses inexorably with time, the reason e for which is unknown14 17. Superficial cartilaginous lesions, viz., structural defects that are confined to the articular-cartilage layer itself and do not penetrate the subchondral-bone plate, represent one of the first overt signs of OA (namely, fibrillation of the carti- e laginous layer and loss of proteoglycans therefrom)18 20. Such lesions do not heal spontaneously21,22. With the lapse of time, they broaden and lengthen, such that what is initially a discrete condi- tion progresses insidiously towards a crippling disease. The reasons why lesions confined to the layer of adult articular cartilage do not heal lie mainly in the nature of this tissue's struc- ture and physiology: it is avascular, aneural and harbours but a sparse population of cells (chondrocytes)23,24, which endure and remain in their allotted positions throughout the life of the host (as is the case for neurons of the central nervous system and for cardiomyocytes). Under metabolically pathological conditions, and in the vicinity of traumatically-induced lesions, the chondrocytes respond but sluggishly to internal and external stimuli; they manifest only a limited proliferative capacity and a reduced e potential for the de-novo synthesis of an extracellular matrix25 27.

Spontaneous repair

From the point of view of its working status, a synovial joint (Fig. 2) can be considered as an organ. The articular-cartilage layer represents but one component of a functional unit, having struc- tural and physiological relationships with the synovial fluid, the synovial membrane, the subchondral-bone plate and the sub- chondral-bone marrow, as well as with the trabecular bone and its vascular system. It is from these associated, highly-vascularized tissue-compartments that we might expect supportive activities for articular-cartilage repair21 e as well as destructive influences28. It was appreciated already at the beginning of the 20th century that if articular-cartilage lesions penetrate the layer of subchondral bone and the bone-marrow spaces e and as a consequence become Fig. 1. Light micrograph of vertically-sectioned articular cartilage, derived from the femoral condyle of a 17-month-old bovine cow, illustrating its subdivision into su- perficial (S), transitional (T), upper-radial (UR), lower-radial (LR) and calcified-cartilage (C) zones; the latter abuts on the subchondral-bone plate (B). 1 mm-thick section, stained with Toluidine Blue. Bar: 200 mm.

The larger joints of the human body, for example, the knee, the hip and the shoulder, are most frequently affected by OA. During the early stages of the disease, a localized pattern of lesioning of the articular-cartilage layer is a characteristic occurrence11. However, even in unlesioned areas of the affected joint, the metabolism of the chondrocytes is disturbed12,13. Once initiated, the lesioning process is progressive, and no prophylactic (medicational) or e interventional measures are at hand to arrest it14 17. Likewise, no biologically-based treatment strategies are available to induce an efficacious healing of the structural defects. This review briefly surveys the biological basis of cartilage repair, as well as current trends in the treatment of articular-cartilage defects, and points out the misconceptional elements in these approaches. Particular emphasis is placed on research activities in the field of cartilage- tissue engineering. The major scientific and practical hurdles that need to be overcome for successful articular-cartilage repair are discussed; so, too, are the shortcomings of repair-evaluation pro- tocols. Suggestions for improvements in the analytical approach are made. Fig. 2. Schematic drawing of a normal synovial joint. 336 E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 imbued with blood bearing diverse chondroprogenitor cells and type of cartilaginous repair tissue and lower incidences of tissue fibrin (which acts as the supportive scaffold of a blood clot) e a degeneration 12 months after surgery than did those who under- limited healing response may ensue26,29. But the repair tissue that went osteotomy alone. However, no difference in the clinical e is laid down by the blood-borne and bone-marrow-derived cells of outcome was observed after 2 (and up to 9) years45 49. diverse nature does not endure for more than a few months17,30,31, The idea of drilling holes through damaged areas of the and even if it persists for a longer period of time, it is deficient in articular-cartilage layer into the subchondral bone-marrow spaces e functional competence32 35. Despite this poor outlook, many of the was conceived by Pridie in 195939,50,51. As aforementioned, the surgical treatment strategies that are in clinical use today rationale behind this concept is to stimulate a spontaneous repair endeavour to foster this spontaneous repair reaction. Interventions reaction. But, perforce, the tissue that is formed is variable in of this kind are often categorized as “bone-marrow-stimulation” composition, structure and durability. The intervention is now e approaches (for review, see27,36 38). performed chiefly in young patients with osteoarthritic conditions, such as osteochondritis dissecans50. The aim is to bring symp- Current surgical strategies tomatic relief and to improve joint functionality52,53 by promoting a resurfacing of the articular layer with a fibrocartilaginous type of Treatment strategies that exploit the spontaneous repair response repair tissue. Clinical reports suggest that patients suffering from by a “stimulation of the bone marrow” generalized arthroses derive the greatest benefits from Pridie drilling54. However, this procedure appears to confer short-term These surgical approaches include abrasion chondroplasty22,29, benefits only, and only in patients of younger age; in the long-run Pridie drilling39 and microfracturing40 (recently refined to micro- (several years after surgery), these beneficial effects are lost55. drilling41,42). The aim of these techniques is to expose the lesioned The microfracturing technique e a modification of the Pridie cartilaginous tissue to the bone-marrow spaces, which, together approach e was elaborated by Steadman et al.40. After the removal with other vicinal compartments (such as the vascular and peri- of the calcified-cartilage layer, multiple holes, 1- to 2-mm in vascular spaces, the bony tissue itself, adipose tissue and the diameter and 3 to 5 mm apart, are created [with either a pointed synovium), are thereby stimulated. In essence, these interventions metallic (ice-pick-like) instrument40 or a narrow-calibre drill41,42] lead to a spontaneous repair response, which is based upon in the bone plate and the bony trabeculae. This process is associated surgically-induced bleeding from the subchondral-bone spaces and with bleeding and blood-clot formation (Fig. 3). The technique has subsequent blood-clot formation. The induction of such a sponta- been recently modified. The improved “microdrilling” version is neous repair response is well known from animal experiments. better controllable, more reproducible and less traumatic41,42,56. However, the ensuing healing response is highly variable and non- The microfracturing technique has thus developed into a “micro- reproducible, and the tissue that is formed is of a fibrous43, unen- Pridie” drilling approach and relies on the same biological princi- during nature26,30. These techniques were introduced several de- ples. The advantage of the latter technique was originally believed cades ago44, at which time no alternative or more efficacious to lie in the avoidance of the thermal necrosis that was thought strategies were available, and in the intervening years a consider- possible with abrasion and drilling. However, this concern has not able body of clinical experience has been gained with them. been confirmed either experimentally or clinically. Microdrilling is Various retrospective clinical investigations appertaining to now performed more widely than any other bone-marrow- e abrasion chondroplasty have been conducted45 48. The success stimulation technique. Albeit so, no prospective clinical trials rates are quite variable49 and depend upon many factors, such as have been undertaken that would either confirm or refute the the patient's age and physical-activity level, the severity of the claimed advantages of this over other bone-marrow-stimulation arthritic condition, and the follow-up period. One relevant clinical techniques. Microdrilling and microfracturing have been advo- trial has been conducted: patients suffering from OA and painful cated chiefly for young patients, in whom good results (improved knee conditions were subjected either to concomitant abrasion joint functionality and relief from pain in 60 to 80% of cases) have e chondroplasty and osteotomy (for realignment and pain relief) or to been reported56 59, probably owing to the larger numbers and the osteotomy alone. Individuals who underwent the combined higher activity-levels of the participating precursor-cell pools in approach manifested significantly higher levels of a “hyaline-like” these individuals, thereby leading to a more exuberant repair

Fig. 3. Schematic representation of the microfracturing technique. The floor of a chondral defect is perforated in several places using either a pointed instrument or a narrow- calibre drill. By this action, the marrow space of the subchondral bone is opened. Blood percolates into the defect e bearing with it local bone-marrow-derived MSCs e and therein forms a haematoma. E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 337 response than in older ones. Since there is some evidence that lessen the burden on health-care systems, which would, in turn, microdrilling and microfracturing are most effective in patients render them more affordable to the average citizen in Western below 40 years of age60, it is questionable whether these tech- societies79. niques would be appropriate for OA-patients, who are usually older, whose spontaneous tissue-repair potential is impoverished, and in Transplantation strategies whom the number and the availability of bone-marrow-derived e stem cells is reduced61 63. Osteochondral autografting (mosaicplasty) With a view to improving the clinical outcome of microdrilling The idea of implanting chondral or osteochondral tissue itself and its reproducibility, this intervention has been combined with within articular-cartilage defects dates back to the beginning of the e the introduction of a thin-layered, blood-absorbing matrix (see last century80 82. And this concept still forms the basis of clinical e e Matrix scaffolds) of either collagen types I and III64,65 or chitosan strategies involving both autografts83 86 and allografts87 89. Of the glycerol phosphate41,66. However, available data afford little evi- autologous osteochondral treatment strategies currently in use, dence of any beneficial effect of this combined approach. The mosaicplasty (Fig. 4) is the one with the longest history. This disappointing results may reflect the relatively high bio- technique is used for large, full-thickness lesions, and involves the incompatibility of the implanted matrical material, which triggers introduction of several or multiple pieces (plugs) of autologous an inflammatory response. Indeed, when this combined approach graft-material into the defect void. The advantage of this treatment is adopted, the defect site is confronted not only with the extrinsic strategy is that it can be performed during a single intervention. matrix but also with the intrinsic one, namely, fibrin, which fills Upon exposure and surveillance of the diseased joint, the ortho- the area after the induction of bleeding, and which likewise triggers paedic surgeon is able to promptly decide from which regions an inflammatory response. Hence, inflammatory reactivity is autologous donor material should be removed and can then pro- augmented by the introduction of an extrinsic matrix. One possible ceed immediately with the autotransplantation (Fig. 4). Mosaic- way to improve the repair result that is achieved in the presence of plasty has for some years been the favoured technique of an extrinsic matrix is to enhance its biocompatibility and to mini- orthopaedic surgeons in Japan and Europe, but is of only limited mize its pro-inflammatory properties. Recent clinical studies that popularity in North America. Indeed, for various reasons (see have been conducted using a collagenous membrane with such below), the approach is now losing credibility among surgeons in e attributes have yielded fairly favourable results67 71. This type of all geographic regions. collagenous matrix is introduced into the defect void after a stim- One of the most serious drawbacks of the technique relates to ulation of the bone marrow by microdrilling. For this combined the destiny of the areas from which the autologous osteochondral approach, clinicians have coined the term autologous matrix- plugs are removed. In taking such tissue, the surgeon is artificially induced chondrogenesis (AMIC), which is a misleading misnomer, creating additional lesions within an already diseased joint (Fig. 4), since the carrier material is not in itself chondrogenic. The advan- and in so doing establishes additional sites of joint degeneration, tages of the AMIC-approach are that it involves no cell- thereby exacerbating rather than assuaging the local pathological transplantation, that it can be performed during a single surgical process. Given the not inconsiderable size of the osteochondral intervention, and that it is cost-effective. Prospective randomized plugs that are excised by the surgeon, the donor sites probably fail clinical trials of a comparative nature are currently underway to to heal. For this reason, some surgeons apply a matrical material to assess the practical usefulness of the AMIC-approach relative to the surgically-created lesions (donor sites), with a view to pro- that of ACI and/or matrix-associated ACI (MACI). moting a spontaneous repair response90. Similarly, we have but a A surer means of achieving an additional improvement in the poor conception of what occurs at the acceptor sites after the clinical outcome of microdrilling-based therapies would be to transplantation of autologous tissue-plugs. The drilling procedures introduce an on-the-site-prepared local population of autologous that are used to remove the osteochondral tissue expose chon- stem cells (either isolated or in the form of tissue-fragments), drocytes within the transplanted plug e as well as those bordering derived, for example, from the synovial membrane or the bone the cavity created at the donor site e to extremely high tempera- marrow. An approach that draws on the on-site preparation of tures and drying conditions, which are liable to impair their vitality. autologous material would involve fewer regulatory hurdles and The osteochondral plugs, once removed, are then press-fitted by circumvent prior laboratory processing, and would, therefore, be hammering into the subchondral-bone bed. This hammering pro- cost-effective. The reproducibility of the repair results could cess probably induces an injurious compression of the transplanted probably be improved by the introduction of a vehicle-borne cartilage91, which could precipitate the immediate death of the growth-factor cocktail to induce the desired chondrogenic differ- chondrocytic population91. Moreover, if the tissue is transferred entiation of the stem cells, which could be conceived as a from a low to a high-weight-bearing region of the joint, it will be e commercially-available, off-the-shelf product72 74, and which subjected to unphysiological conditions of mechanical loading for would have to be applied together with a functional or structural which it was not conceived, thereby accelerating its degeneration. barrier75,76 to prevent the up-growth of underlying bone into the The insertion of several serried plugs within the defect void, cartilaginous compartment. A concept that is based on the use of without taking measures to promote knitting between them, might fragments of synovial membrane would be the one most likely to also give rise to complications: the absence of structural bonding meet with success, since stem cells of synovial origin have a higher around the entire circumference of each plug will certainly pose chondrogenic potential than those derived from either the bone nutritional as well as functional problems from the outset, and marrow, the periosteum, adipose tissue or muscle, at least in vitro77. these will tend to prompt tissue degeneration (see Ref. 27). Only a Moreover, it would even be advantageous to introduce the cells in few attempts have so far been made to clarify these issues. Hence, the form of tissue-fragments rather than as isolates, since, when although mosaicplasty is still used, there exists no sound scientific supported by their physiological scaffold, their responsiveness to basis for the contention that patients subjected to this treatment e the applied chondrogenic differentiation factor(s) would be better strategy benefit in the long run92 98. assured78. New treatment strategies should be preferably conceived from the onset as single surgical interventions or Osteochondral allografting arthroscopic approaches e with a view to clinical practicability and For many decades now, allogeneic osteochondral grafts have to reduce the risk of infection or other complications, as well as to been used for the filling of articular-cartilage defects. The approach 338 E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350

Fig. 4. Schematic representation of the principle of mosaicplasty. Cylindrical plugs of osteochondral tissue are drilled from a non-weight-bearing area of the knee joint (using a special surgical instrument) and then press-fitted, side-by-side, within the damaged zone. The number of plugs that are removed will depend upon the surface diameter of the acceptor site. The denuded donor sites are either filled with osteochondral tissue that is removed from the damaged acceptor site during its surgical preparation for the plugs, or left empty. When viewed from above, the fitted plugs form a mosaic of circular profiles that are interspersed with empty spaces; hence the name “mosaicplasty”.

does not attempt to induce a cartilage-repair response, but repre- fact the brainchild of Grande et al., who tested it in a preclinical sents a means of substituting failed or lost tissue with healthy study with rabbits in 1989114. According to the ACI-technique, a articular cartilage, which is usually derived from cadavers80. biopsy of healthy articular cartilage is first arthroscopically har- The biological rationale for this approach is based on the vested from a low- or a non-load-bearing location of the diseased knowledge that large osteochondral defects do not heal sponta- joint. The cartilaginous tissue is then enzymatically digested to neously and that it is extremely difficult to elaborate a suitable release the chondrocytes, which are subsequently expanded in stimulatory system to promote effective repair in a compartment- culture, thereby generating more than 10 million cells from the few specific manner within the cartilaginous and the osseous carrels. hundred thousand that are contained within the biopsy. A sus- It has thus been deemed simpler to replace the lost tissue with pension of the expanded autologous chondrocytes is introduced e cadaverous osteochondral allografts99 101. Immunological prob- directly into the defect, beneath a surgically-sutured periosteal lems are inevitably to be counted amongst the drawbacks of this flap, which is usually removed from the medial tibia. Systems technique102,103. Nevertheless, patients with large osteochondral involving the application of freely-suspended cells are technically defects, such as those generated by tumour resection, osteonec- more complex than those in which matrix-embedded cells are rosis, extensive trauma, widespread OA or osteochondritis dis- grafted, since the careful placement and suturing of periosteal secans, benefit greatly from this treatment strategy104,105. flaps (or other types of material with a comparable function) are Clinical experience with this therapeutic option has been sur- required to ensure their retention and optimal integration with the prisingly good, immunological reactions being apparently less surrounding articular cartilage. Furthermore, the postoperative extensive in humans than in experimental animals103,106,107. Hu- delamination of such flaps e which can and does occur, even after man osteochondral allografts also survive for longer periods (some suturing115 e must be prevented by a temporary immobilization years), even after freezing or lyophilization87,88,102,108. Clinical (partial or complete) of the joint. More recently, a matrix- success rates have been reported to range from 65% to 85%, even associated variant of the ACI-approach (the so-called MACI-tech- e after monitoring periods of up to 10 years100,109 111. The major nique) was introduced116. According to this MACI-technique, the drawbacks that are associated with the implementation of this autologous chondrocytes are embedded within a matrix (e.g., methodology in clinical practice include the scarcity of fresh donor collagen or hyaluronan) rather than suspended in a fluid, thereby material and the storage of frozen tissue112. The small but ever- obviating the need for a periosteal flap. However, the use of a present risk of disease transmission must also be borne in mind111. matrix introduces a new set of problems, such as the issues of bioincompatibility, inflammation and degradation (see Trans- Autologous-chondrocyte implantation (ACI) plantation strategies, and Refs. 27,117). The ideal carrier118 has not yet been developed. Albeit so, this is still a goal towards which When the ACI-technique (Fig. 5) was introduced on the clinical researchers aspire, and efforts in this direction have not been scene by Brittberg et al. in 1994113, it took the orthopaedic world by thwarted by either the slowness of the progress or the set-backs of storm, generating much hope and enthusiasm. The concept was in failure. E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 339

Fig. 5. Schematic representation of the ACI-technique. A fragment of articular cartilage is removed from the periphery of the joint that bears the lesion. This fragment of tissue is digested to release the chondrocytes, which are expanded in vitro for 11 to 21 days. A periosteal flap is then removed from the medial tibia and sutured over the lesion. A suspension of the expanded chondrocytes is injected beneath the flap into the lesion.

A considerable disadvantage of ACI-based strategies is that Almost unbelievably, this possibility has not been excluded in the surgical intervention is required on two separate occasions, which published studies by the establishment of the appropriate controls is a costly process necessitating long-term patient planning. The [viz., an “empty” defect (negative control): no injection of chon- creation of additional lesions within the joint during the harvesting drocytes beneath a periosteal flap114,116; and a defect filled with of healthy cartilage is also a drawback. Although only a small biopsy another cell type (positive control), e.g., fibroblasts]. is taken from a non-weight-bearing area of the joint, some in- Neither microfracturing/microdrilling nor ACI is clinically vestigators claim that this may heighten the risk for the develop- effective in patients over 50 years of age, probably because the ment of OA119,120. It has also been suggested that the chondrocytes bone-marrow-derived (mesenchymal) stem cells (microfracturing could be removed from osteoarthritic rather than from normal technique) and the transplanted autologous chondrocytes (ACI- articular cartilage, or even from other tissue sources121,122. technique) suffer age-related losses in their potential to proliferate Although the ACI-technique is still highly popular, its superiority and differentiate125. Hence, especially for elderly persons, who are to simpler interventions, such as microfracturing or microdrilling, particularly prone to osteoarthritic lesioning of the articular- has not been established. Knutsen et al.123 have published the re- cartilage layer, there exists an undisputed need for an effective sults of a prospective randomized clinical trial in which patients cell-based repair strategy. We have to face the bleak fact that very with circumscribed cartilaginous lesions in the femoral condyle little progress in this area has been made since the bone-marrow- were treated either conventionally, namely, by microfracturing, or stimulation technique e of which microfracturing/microdrilling is a according to the ACI-technique. The findings of this 5-year trial variant e was introduced by Pridie in the 1950s44. However, one revealed the latter strategy to confer no advantage over the con- source of comfort is that cell-based cartilage-repair strategies, such ventional one. Although the repair tissue that was evident 1 year as the ACI-technique, do not elicit worse healing results than those after treatment according to the ACI-technique was reported to be that are based on a stimulation of the bone marrow. A refinement of structurally superior to that laid down after microfracturing, the the ACI-technique has been recently introduced, which involves a clinical outcome was the same124. An ongoing long-term follow-up selection from the harvested cells of specific sub-populations of analysis of the same cohort of patients has confirmed the earlier (characterized) chondrocytes with particularly high chondrogenic (5-year) findings: 15 years after treatment by microfracturing or potentials. However, 2 years after treatment according to the according to the ACI-technique, the clinical results were the same in refined ACI-technique, the clinical benefits were reported to be only each group, which argues in favour of the less traumatic, less risky incremental126. And 5 years after treatment by microfracturing or and less costly microfracturing approach (Gunnar Knutsen: per- according to the refined ACI-treatment, the clinical outcome was sonal communication). A possible explanation for these short- and ultimately the same in each patient-group127, despite some differ- long-term findings is, that from a biological and biomechanistic ences during the follow-up course128. point of view, the treatments are basically the same. When the ACI- Given the high degree of technological finesse that is now technique is performed, the floor and the walls of the defect are achievable, and against the backdrop of knowledge and experience usually “neatened” by shaving, which induces local bleeding and a that have been gained over the past 60 years with Pridie drilling spontaneous repair response from the bone-marrow spaces. and over the last 20 to 30 years with the ACI-approach [as well as 340 E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 with its variations/modifications (e.g., MACI)], both strategies are sections, the key constituents of a tissue-engineering-based accessible to improvement. As aforementioned, in an attempt to approach to cartilage repair are described. improve the clinical outcome of microdrilling and its reproduc- ibility, the approach has been combined with the introduction of a Cell sources thin-layered, blood-absorbing matrix (enhanced microfracturing) of either collagen types I and III64,65, “manipulated” collagen (as in Success in tissue-engineering-based strategies would obviate the AMIC67) or chitosan glycerol phosphate41,66. The available data need for the transplantation of cartilaginous tissue. And if appro- afford evidence that the clinical results are reproducible68,71, and, priate precursor-cell pools could be identified, then the numerous possibly, that the long-term outcome (compared to the conven- problems that are associated with the use of donor (allogeneic) tional technique) may be improved70. As heretofore mentioned, the material would be circumvented. Precursor-cell pools of diverse introduction of a local population of autologous stem cells (e.g., in tissue origin are known to exist within adult mammalian organisms, the form of small tissue-fragments, which could be derived from a each of which has the potential to differentiate into cartilage- local source, such as the synovium) would probably be a fruitful producing chondrocytes and which can be used for trans- route to pursue in the future. However, the currently practiced plantation purposes. And in some instances, the tissue of origin has approaches, which involve the transplantation of either fragments the potential for self-repair after the removal of not insubstantial e of juvenile allogeneic cartilage129 131 or pieces of adult autologous quantities of material. The tissue sources include the cambial layers e articular cartilage129,132 134, are based on the false premiss that the of the perichondrium and the periosteum, the bone-marrow stroma, e cells can migrate out of the tissue. Tissue-embedded chondrocytes the synovial membrane, muscle, fat143 145 and adult cartilage itself e are incapable of migration; they can undergo only indirect trans- (autologous or allogeneic chondrocytes146 148). Perinatal cells149,as location via a directional resorption of the surrounding matrical well as foetal or embryonic stem cells and chondroblasts, can also be coat and its neoformation135. Migration is possible only if the cells employed150. The search for suitable and hitherto untested types e undergo dedifferentiation (which occurs during the late stages of continues unabated151 153. Due to the limited availability of autol- osteoarthritis) and if the pericellular and territorial matrical com- ogous chondrocytes, the focus is now more on allogeneic sources. e partments are destroyed by excessive catabolic activity136 138. However, the use of allogeneic cells raises other problematic issues, Clinical studies that have dealt with these approaches129,133 suffer such as bioincompatibility and immunogenicity142,152. from fundamental shortcomings, which include insufficient Mesenchymal stem cells (MSCs) are now frequently used numbers of patients, the absence of a basis for comparison (e.g., experimentally for cartilage-tissue engineering, owing to their ca- e sham surgery), and the failure to instigate random-selection pro- pacity for self-renewal and their accessibility143 145. Amongst the cedures. The data that have been gleaned from such trials serve various tissue sources, bone-marrow-derived MSCs have been the only to demonstrate that the interventions can be safely imple- most intensively investigated, both in vitro and in vivo, since the mented; they do not permit the drawing of comparative conclu- time of their original isolation by Friedenstein in 1966154. Bone- sions. The same arguments hold true for clinical studies that have marrow-derived MSCs evince a high capacity for chondrogenesis evaluated the efficacy of approaches involving the application of a only in the presence of appropriate stimulation factors78,155,156.Ina e bone-marrow-aspiration concentrate (BMAC)139 141 or of platelet- clinical setting, Kuroda et al.157 treated a large full-thickness artic- rich-plasma (PRP), either alone or in combination with ular-cartilage defect within the femoral condyle of an athlete with BMAC140,141. PRP- and BMAC-based strategies are classical examples autologous bone-marrow-derived stromal cells. The bone marrow of empirical, “alchemistic” approaches to the engineering of carti- was aspirated from the patient's iliac crest 4 weeks before surgery laginous tissue. Using the former approach (PRP), an ill-defined and the cells were expanded in culture after their separation from broth of multifarious growth factors at unknown concentrations the erythrocytes. The undifferentiated cells were then embedded and with diverse e antagonistic as well as synergistic e workings is within a collagenous gel, which was transferred to the articular- administered; and using the latter (BMAC), a similarly ill-defined cartilage defect and covered with an autologous periosteal flap. soup of diverse cells in variable numbers is applied. Owing to Although the knee-outcome scores improved with time, biopsies inter-individual variations in the compositions of these pot-pourris, revealed the presence of fibro-, not of hyaline, cartilage. In this the exerted effects will be likewise variable and thus irreproduc- study, no exogenous agents were applied to guide the course of ible; and even if these are positively rated, an identification of the differentiation. This fact suggests that the formation of a hyaline key-player(s) is impossible. type of repair cartilage by MSCs requires the presence of appro- priate stimulation factors (see Signalling molecules), which may be Current research activities relating to tissue-engineering- locally absent, or present at insufficiently high levels to guide the based strategies process of differentiation in the desired direction. Although MSCs have been claimed by some investigators to furnish their own Many more research teams around the world are now moving supply of growth factors158,159 and to be conferred with the ca- away from an overly empirical approach to cartilage repair, which pacity for immunomodulation160, experimental experience gained was hitherto the norm, towards a more biologically rational one. over the past 20 years in the field of cartilage repair has revealed Current activities tend to focus on the elaboration of novel tissue- these activities e if indeed they exist e to be too low to support an engineering-based strategies142. Tissue engineering can be defined efficacious healing response. as the art of reconstituting mammalian tissues, both structurally and One of the limitations of bone-marrow-derived MSCs is that functionally. Such reconstructive processes can be conducted (1) their chondrogenic potential declines with increasing patient entirely in vitro, (2) initially in vitro and then in vivo (in situ), or (3) age125. MSCs that are derived from the synovium do not suffer from entirely in vivo. this drawback161. Furthermore, the chondrogenic potential of MSCs Three key constituents usually form the building blocks of a originating from the synovium appears to be superior to that of tissue-engineering-based approach: a matrix scaffold, cells, and MSCs originating from either the bone marrow, the periosteum, signalling molecules (such as growth factors or genes). Most of the adipose tissue or muscle, at least in vitro77. However, as yet, we are experience so far gained with these techniques has been derived still in the dark as to which tissue source of MSCs is ideal for the from in-vitro studies, although experiments with laboratory ani- clinical repair of articular cartilage, since convincing in-vivo data mals and humans have also been conducted. In the following have not been forthcoming to date. E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 341

Table I programming of joint-surface shape or of locational differences in Chemical classes of matrix structure, composition and thickness23,24, is lacking. Knowledge of 1. Protein-based polymers this kind is indispensable for the fruitful development of more Fibrin sophisticated epigenetic and higher-level approaches to cartilage Collagen repair. Only against a background of improved knowledge and Gelatine understanding can we hope to engineer a mechanically-competent 2. Carbohydrate-based polymers Polylactic acid and durable type of articular cartilage. At present, research in this Polyglycolic acid area has attained a hyatal status and is moving in circles rather than Hyaluronan forward. Agarose Alginate Chitosan Matrix scaffolds 3. Artificial polymers Dacron (polyethylene terephthalate) The use of matrices as cell-carriers in cartilage-repair strategies fl fl Te on (polytetra uoroethylene) dates back to the 1960s163. Many different types of matrix have fi Carbon bres e Polyesterurethane been tested not only in vitro but also in experimental animals and Polybutyric acid human patients e for their capacity to facilitate or promote Polyethylmethacrylate articular-cartilage repair. These matrices can be broadly categorized Hydroxyapatite according to their chemical nature into protein-based polymers, 4. Within/between classes carbohydrate-based ones, artificial materials, and combinations of Cross-linkage Chemical modulations these (Table I). Even when a naked matrix (viz., one that carries no Geometrical modifications (to produce fibrillar forms or foams) cells or signalling substances) is deposited within an articular- Matrix combinations cartilage defect, it must be considered as an information-carrying Reproduced e in a slightly modified form e with permission from30. device: its three-dimensional structure will, according to its peculiar geometrical configuration, specifically guide, or at least influence, the process of tissue growth, whilst the chemical and Irrespective of the type of stem cell that is employed, tremen- physical properties of its surface will impact cell attachment and dous hurdles must still be surmounted before the goal of cartilage the binding of growth factors in the niche environment. Hence, as regeneration can be achieved. Our knowledge of the regulatory and an “active biologic”, such a naked matrix will elicit a biological signalling pathways that are implicated in the biology of stem cells response within a bodily tissue-compartment, which may or may generally, and more especially that govern their commitment to not be desirable, but which cannot be prevented unless specific differentiate along a specific course and to form a specific type of modulations of an appropriate nature are made to guide the pro- cartilaginous tissue, namely, fibro-, hyaline or elastic, is still very cess in the desired direction [e.g., by means of AMIC (see discussion incomplete. Information appertaining to the temporal course of above)]. The basic requirements of a matrix to be used in a chondrogenic differentiation and its regulation is also lacking. With biological environment are summarized in Table II (see also respect to induced pluripotent stem (iPS) cells162, for example, it Refs. 27,117,118). still remains to be ascertained whether the point in time at which Although some of the listed materials are already in clinical they are induced to undergo committed differentiation along a use164, most of them, and especially the synthetic ones, are still specific course is or is not programmed into their genetic appa- being tested in preclinical trials165. Moreover, using scaffolding ratus; and, if so, whether the “age”, viz., maturation state, of the materials that have been applied for awhile in the field of cartilage newly-formed cells and tissues matches that of the donor. The same repair, such as collagen166, fibrin167, hyaluronan168,169 and poly- questions arise also when dealing with foetal, embryonic or adult lactate170, as well as those that have appeared on the scene more stem cells. Moreover, we are still in the dark as to whether the recently, attempts are now being made either to produce them in tissue-differentiation and growth processes that ontogenetically nanofibrillar forms171, to enhance their porosity172, or to manu- span decades in time can indeed be speeded up therapeutically so facture them in a state that is conducive to injection173. The com- as to be completed within the matter of a few weeks only. mon aim of these endeavours is usually to create an absorbable, Furthermore, although our knowledge of the human genetic code is biomimetic material174, even when using synthetic polymers, such now fairly complete, information relating, for example, to the as polydioxanone175.

Table II Matrix requirements

Matrix property Biological basis

1. Porosity Cell migration 2. Carrier Lodgement and release of signalling substances 3. Adhesion Cell attachment 4. Biodegradability Physiological remodelling 5. Volume stability Smooth surface contour of repair tissue flush with that of native articular cartilage 6. Biocompatibility Good contact with the native-tissue compartment 7. Bonding Enhances interfacial integration between collagenous fibrils in repair- and native-tissue compartments 8. Internal cohesiveness Prevention of matrix outflow 9. Elasticity Resiliency during and following dynamic or static deformation 10. Structural anisotropy Promotion of native anisotropic tissue organization

Matrix properties specific to the mode of surgical application A. Fluid state during application with subsequent solidification in situ Arthroscopic implantation B. Stiff and amenable to press-fitting Arthrotomy (open surgery of a joint)

Reproduced e in a slightly modified form e with permission from30. 342 E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350

More recently, it has become the vogue to use biphasic scaf- There are three possible approaches to the engineering of folds176,177. These bilayers consist of two different materials: an articular cartilage. According to the first, the repair tissue is engi- upper one that is deemed to be conducive to chondrogenesis and neered completely in vitro, the fully-differentiated construct being which is destined for the cartilaginous compartment of osteochon- then implanted within the defect void. The advantage of this dral defects, and a lower one of a ceramic-like substance, which can approach lies in the fact that cell metabolism and differentiation be press-fitted as a plug into the subchondral bone176. Apart from the are subject to better control in vitro than in vivo, provided that the obvious issues that must be addressed when testing a scaffold, such appropriate bioreactor systems195,196, growth factors and delivery as biocompatibility, biodegradability and toxicity, its integration systems are available. Its drawbacks are, that the problems asso- with native cartilage is also a troublesome problem, and one that has ciated with the integration and the mechanical fixation of the not yet been overcome178. During the past decade, no matrices with repair tissue cannot be readily anticipated and solved. Moreover, basically new chemical features have been inaugurated. However, the simulated mechanical-loading conditions of the extracellular physical or chemical modifications to existing materials have been matrix may not be ideally suited to the specific needs of the pro- effected, with a view to enhancing the efficacy of clinically- spective repair site. In addition, biocompatibility and immunolog- established methodologies, such as microfracturing/micro- ical problems are frequently associated even with this in-vitro e drilling41,64 66, by AMIC68,70, or ACI (by MACI)116, or, in the context of approach and in the absence of a scaffold197. Furthermore, the more experimental tissue-engineering-based approaches, with the natural curvature of the joint surface poses a challenge to the aim of guiding cartilage-formation in a spatially-defined manner process of press-fitment whereby the engineered construct is [three-dimensional bioprinting (see discussion below)]. The dearth introduced into the defect void. of genuine innovation in this field partially reflects the unfavourable The second approach, which is more frequently adopted, aims to investment climate that prevails in the commercial world, which engineer only the basic building block, namely, a matrix scaffold encourages low-cost, incremental improvements in existing stra- containing a homogeneous population of cells, and signalling mol- tegies rather than revolutionary novelty. This is a regrettable ecules that are entrapped within an appropriate delivery system. circumstance, since it is precisely this flux of ingenuity that is now The vehicle-bound signalling molecules ensure that the desired needed to push research in a really fruitful direction. However, one differentiation process takes place in a controlled and timely positive aspect of this dilemmatic situation is that any newly- manner in vivo and that chondrocytic activity within the repair conceived therapeutic principle must now be not only of a truly tissue is sustained. According to this approach, the differentiation impressive nature but also backed up by rigorous experimentation and the remodelling of the repair tissue occur in vivo under physi- before any willingness to invest in clinical trials can be expected. ological conditions of mechanical loading. Repair tissue that is formed in situ is more likely to adhere to and integrate with native Signalling molecules articular cartilage than is that produced in vitro, and it will also adapt naturally to the contour of the synovial joint, provided that appro- One of the trends now foremost in the field of tissue engineering priate measures are taken to promote the bonding of the implant e relates to the elaboration of constructs that consist not only of a that has been generated in culture198 200. One of the disadvantages matrix and cells but also of signalling molecules to specifically of this “second” approach is that cell activity is more difficult to guide the course of differentiation in the desired direction. In most control on a long-term basis. Moreover, appropriate measures must cartilage-engineering strategies, the chosen cell type is applied be taken to avoid “contamination” from cells and signalling sub- together with free or appropriately-encapsulated growth factors. stances that are involved in the spontaneous healing response, since The efficacy of various growth factors to induce chondrogenesis has the tissue thereby formed would compromise the quality and the been evaluated mainly in vitro and to only a limited degree in vivo mechanical competence of the final repair-composite. (for reviews, see Refs. 179e183). In-vitro experiments have involved The third approach involves the direct application of exogenous various combinations of matrix, cell type and growth factors. These growth factors (entrapped within a matrix) to the defect site; these include collagenous matrices containing bone-marrow-derived then stimulate the intrinsic formation of cartilaginous tissue in stromal cells and transforming growth factor (TGF)-ß1184,185, situ72. An appropriate matrix is first introduced into the defect to agarose matrices containing chondrocytes and fibroblast growth define the space that is to be repaired. The laying of this physical factor (FGF)-2186, fibrinous matrices containing chondrocytes and “track” is necessary to guide the movements of the intrinsic pre- insulin-like growth factor (IGF)-1187, and polylactatous matrices cursor cells, which have a limited spatial awareness21. The growth containing perichondrial cells and TGF-ß1188. In some instances, factors are usually introduced into the matrix in two different states: MSCs have been cultured in the absence of a matrix (viz., under as a freely-soluble agent e to stimulate the immediate recruitment scaffold-free conditions), namely, as high-density pellets (or ag- of the intrinsic population of precursor cells from their site of origin, gregates)183,189,190. Under such conditions, not only growth factors, their migration into the defect area, and their proliferation therein21 but also dexamethasone, a synthetic glucocorticoid, is frequently e and in a vehicle-bound form for gradual delivery at a steady rate applied. However, this agent can elicit undesired effects in a that is sustained for several weeks e to stimulate the chondrogenic manner that depends upon the source of the cultured cells and the differentiation of the defect-filling population of cells72. Hence, an absence or presence of the natural (physiological) extracellular intelligent, internally-programmed matrix is applied to the defect. matrix78,155,156. Successful testing in vivo has been achieved using The freely-soluble signalling agent that is applied is usually IGF-1, equine191 and mature miniature-pig models72. The study that was bFGF or a TGF-b (at low concentration). As aforementioned, the conducted using the latter model has served to show how different growth factor in this form stimulates the recruitment of precursor the in-vivo situation may be from the in-vitro one. Under in-vitro cells from the synovium21, their migration into the defect space, and conditions, various types of growth factor have been shown to their subsequent proliferation therein. The vehicle-bound signalling support chondrogenic activities192, whereas in vivo, principally only agent that is applied to induce the chondrogenic differentiation of those of the TGF-b superfamily are effective193. A recently- the defect-filling population of cells is usually a member of the TGF- discovered compound, namely, kartogenin194, is now attracting b superfamily, for example, bone morphogenetic protein (BMP)-2, some attention. However, this molecule appears to exert a gener- BMP-9, BMP-13 or TGFb1 (at high concentration). Various drug- alized anabolic effect on chondrocytes rather than being specif- delivery systems have been employed, including microspheres, li- ically chondrogenic. posomes, microparticles and fibrillar networks. The principle E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 343 underlying this approach was first published by our group in In this review, suggestions for fruitful directions of pursuit in the 1991201. A full description of the strategy appeared in the literature future have been made. But what is now needed is a revolutionary in 199621 and then, in an optimized form, in 200172. The approach turnaround in our conception of cartilage repair and a more thor- has since been summarized in several publications27,202. The prin- ough and systematic approach to its evaluation. We should not ciple has been recently confirmed by an independent group of in- stagnate, but break free of the bounds of our comfort zone as vestigators203 (2010) using TGF-b3 as the chemotactic and represented by the old well-worn ruts e the trusted, tried and chondrogenic differentiation agent, a hydrogel as the drug-release tested tracks of yore e the pursuit of which can contribute no more system, and three-dimensional-printed poly-ε-caprolactone-hy- than incremental or cosmetic information to the body of existing droxyapatite as the matrix. Unfortunately, the authors chose not to data that has accumulated over the past decade. cite the original pioneering work on this topic and coveted the idea A fundamental change in the general attitude of scientists, PhD- as a new one. Actually, the novelty of the study lay in the use of poly- students and supervisors towards the evaluation of cartilage repair ε-caprolactone-hydroxyapatite as a three-dimensionally-printed is also called for. In many laboratories, the analytical process is matrix. A matrix of this kind is implemented with a view to guiding conducted on an elementary e almost routine e and insufficiently tissue formation in a spatially-defined manner. As appertaining to discriminative level. Nowadays, repair cartilage is all too commonly articular cartilage, the aim is to reproduce the anisotropic organi- presented as a tissue that contains lacuna-mantled cells (chon- zation of the chondrocytes and their extracellular matrix that is so drocytes), that stains histochemically with a cationic dye (such as characteristic of the mature tissue and that is indispensable for its Toluidine Blue or Safranin O), and that manifests immunohisto- mechanical competence. Although three-dimensional bioprinting is chemical reactivity for type-II collagen. Attempts to quantify the an old technique (for a review of the topic, see Ref. 204), it has characteristics of the tissue are confined to parameters that insuf- recently undergone a revival: owing to technological advancements, ficiently describe the true nature of articular cartilage. These pa- sophisticated materials can nowadays be manufactured with rameters are gauged using subjective and biased scoring systems, greater facility and more cheaply than heretofore. such as those propounded by Mankin215, O'Driscoll216 and the The material of which the matrix is formed needs to be suffi- ICRS217. The parameters that are evaluated using these tools afford ciently porous to allow the infiltration of cells27,201 and also biode- no information relating to the maturation status of the repair gradable. Ideally, it should also have a well-defined anisotropic cartilage, viz., whether it is of a foetal, ontogenetically-transient, organization, which forces the cells to align themselves into vertical early postnatal or adult type; nor is any information gleaned as to columns during the process of remodelling205 and which is whether the repair cartilage is of a joint-specific type or not. important for the mechanical competence of the tissue as a whole, Similarly, these rudimentary evaluations yield no information as aforementioned. In this context, the three-dimensionally-printed respecting the metabolic status of the tissue, viz., whether the matrix that represented the sole novelty of the study by Lee et al.203 anabolic and the catabolic activities are in a state of equilibrium or may be advantagenous. not. Very often, the repair cartilage is sweepingly designated as One problem appertaining to the chondrogenic differentiation being “hyaline-like” without adequate supportive evidence. The of the defect-filling population of cells that we have recently repair cartilage needs to be subjected to a rigorous analysis: its addressed relates to the timely arrestation of the process prior to maturation status should be defined; so, too, should its architec- the stage of terminal hypertrophy and matrix mineralization73. This tonic and ultrastructural features, including the degree of anisot- undesired event can be avoided by the addition of TGF-b1 at a low ropy. These and other structural characteristics should be concentration73. Other investigators have achieved similar results quantified morphometrically, not using subjective scoring systems. e using alternative agents206 212. Another drawback of scoring-system-based approaches is that Consequently, to achieve optimal cartilage-repair results, mini- they attempt to assess too many parameters, whereas ideally, three mally three sets of growth-factor-stimulated “activities” need to be to maximally four pertinent ones should be evaluated. The impact e embraced in the applied cocktail of signalling agents72 74. This ne- of a repair strategy on the individual parameters can vary greatly cessity, which is not always recognized (cf203), renders the approach (ranging from positive to negative), and yet, in the end, an average unattractive to cost-conscious commercial parties and/or investors. score for all is calculated. Hence, there is the danger that positive Principally two novel growth-factor-based therapeutic strate- findings for some parameters will be “diluted” by negative effects gies are now at the conceptual stage of development: the afore- on others, and, in extreme cases, that falsely-negative results will mentioned one, which is designed to recruit cells with be generated; the reverse situation also holds true. Unfortunately, chondrogenic potential from a local intrinsic source, such as the one cannot escape from the reality that scoring-system-based ap- synovial membrane21,72,203; and a second approach, which involves proaches are subjective and biased, since they attempt to describe the injection into the defect site of MSCs that have been transfected the situation that appertains within the volume of a defect void with genes encoding the factors necessary for chondroprogenitor from two-dimensional images of histological sections without commitment213,214. Both approaches are still at a preliminary stage respecting the laws of random sampling and stereology. And it is of development; exhaustive short- and long-term preclinical precisely these laws that a morphometric analysis does respect. The studies79 are requisite prior to the launching of clinical trials. authors feel the necessity of dissuading investigators from imple- menting a scoring-system-based approach, which may not “go Scientific evaluation down well” with those who have used such regimes for many years. However, “a man should never be ashamed to own he has been After perusing this review article, the reader will no doubt be wrong, which is but saying, in other words, that he is wiser today left with the impression that in the area of cartilage repair, research than he was yesterday” (Alexander Pope: Miscellanies). In- activity over the past 10 years or so has turned around in circles, not vestigators should not be daunted by the prospect of undertaking a moved forward in excitingly new and fruitful directions e which is morphometric analysis. Once the basic principles of the approach a regrettable situation for hopeful patients. The circumstance is the have been grasped (for a simple introduction to these, see Ref. 218), less excusable in so far as the technological advancements that have all that is needed for an objective and unbiased evaluation of a few been made during this time span have greatly aided the probing of pertinent parameters are correctly-sampled tissue-sections and an many facets of cartilage biology, thereby permitting an enrichment appropriate grid-system. The investigator's task is then a simple of knowledge and understanding in the targeted disciplines. “counting” one; moreover, he or she is not burdened with the 344 E.B. Hunziker et al. / Osteoarthritis and Cartilage 23 (2015) 334e350 responsibility of “judgement”, which is the case when a scoring Jakob and Gunnar Knutsen for their valuable input respecting the system is implemented. critical analysis of clinical studies. In addition to structural data, information appertaining to the post-translational metabolic status of the chondrocytes should be derived by monitoring the gene-expression levels of representative References anabolic and catabolic markers. The conception of truly novel therapeutic approaches to carti- 1. Hunziker EB. Articular cartilage structure in humans and lage repair is greatly encouraged. But all the ingenuity in the world experimental animals. In: Kuettner KE, Schleyerbach R, will be as wind-blown chaff unless we make a stand against the Peyron JG, Hascall VC, Eds. Articular Cartilage and Osteoar- quick-and-easy attitude that is now sadly enough the brand of thritis. New York: Raven Press; 1992:183e99. modern times. 2. 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