Surgical osteochondral defect repair in the horse—a matter of form or function?

Abstract Focal cartilaginous and osteochondral lesions can have traumatic or chondropathic degenerative origin. The fibrocartilaginous repair tissue that forms naturally, eventually undergoes fibrillation and degeneration leading to further disruption of joint homeostasis. Both types of lesion will therefore eventually lead to activity‐related pain, swelling and decreased mobility and will frequently progress to osteoarthritis. Most attempts at realising cartilage regeneration have so far resulted in cartilage repair (and not regeneration). The aim of this article was to review experimental research on surgical cartilage restoration techniques performed so far in equine models. Currently available surgical options for treatment of osteochondral lesions in the horse are summarised. The experimental validity of equine experimental models is addressed and finally possible avenues for further research are discussed.


| INTRODUC TI ON
Focal cartilaginous and osteochondral lesions can have traumatic or chondropathic degenerative origin. The fibrocartilaginous repair tissue that forms naturally, eventually undergoes fibrillation and degeneration leading to further disruption of joint homeostasis. 1 Both types of lesion will therefore ultimately lead to activity-related pain, joint effusion and decreased mobility, frequently progressing to osteoarthritis. 2 William Hunter's statement made in 1743 of articular cartilage being a tissue that 'when destroyed, it is never recovered' 3 is still applicable. In terms of 'recovery' of tissues, a distinction should be made between tissue regeneration and tissue repair.
Regeneration refers to healing in which there is regrowth of tissue towards the original, normal state. In repair, there is a combination of regeneration and replacement by laying down connective tissue, mostly referred to as scarring. 4 Most, if not all, attempts at realising cartilage regeneration have so far resulted in cartilage repair, not far from what endogenous repair would achieve in a joint.
Compared to other animal models, articular cartilage thickness and subchondral bone thickness in the stifle of adult horses most closely approximates that of the human knee 5,6 ( Figure 1). The horse is an athlete that suffers from similar debilitating cartilage lesions as human patients and therefore, in addition to being a patient in its own right, the horse is a model for the human patient. Because defects of relatively large size can be made experimentally in the horse, more outcome parameters (arthroscopic re-evaluation, histological assessment, biomechanical testing, diagnostic imaging, biochemical analysis) can be measured with each repair response than is possible in other animal models. 7 Based on two fundamental studies on spontaneous cartilage healing of experimental lesions in the equine stifle, the clinically relevant size for created osteochondral lesions has been determined to be 9 mm in diameter. 8,9 In the medial femoral condyle this corresponds to 15%-20% of the weightbearing surface. 10 For the intercarpal joint a more recent study defined 4 mm in diameter as the critical size for osteochondral lesions. 11 These critical defect sizes refer to filling of the defect with repair tissue, not to tissue regeneration.
The aim of this article was to review high-quality experimental research on surgical cartilage restoration techniques in the horse.
Currently available surgical options for treatment of osteochondral lesions in the horse and their experimental validity are summarised.
Applicability to human patients and the validity of equine models is addressed and finally possible avenues for further research are discussed.

| E XPERIMENTAL S TUD IE S ON SURG IC AL TECHNIQUE S
The experimental studies discussed below are summarised in Table S1.

| Microfracture
Marrow stimulation techniques, in particular microfracture, are routinely used for full-thickness defects with an intact subchondral bone plate in horses. 12 This procedure is believed to stimulate endogenous cartilage repair and to facilitate influx of stem cells and growth factors that originate from beneath the subchondral bone plate. Three basic research studies on microfracture in the horse in the medial femoral condyle and in the radial bone of the carpus have been performed, but only one is a long-term study (12 months). Lesions treated with microfracture showed more defect filling when compared with no treatment in terms of quantity of repair tissue. 10,13,14 Histologically, composition of repair tissue, including the relative presence of collagen type 2, was not different between lesions treated with microfracture and untreated lesions. Functionality in terms of biomechanical strength of the repair tissue was not assessed in any of these studies. In the human field, microfracture has been questioned, because studies supporting effectiveness are mainly derived from case series and there are few randomised trials. 15 A large systematic review on microfracture for the treatment of osteochondral defects in the knee in human patients showed that in most cases clinical outcomes improved with microfracture at short-term, but in some studies and over the longer term these effects were not sustained. 16 One of the negative outcomes appears to be the formation of intralesional osteophytes. [17][18][19][20] This might represent further degeneration of repair fibrocartilage triggering a reactivation of the endochondral ossification mechanism once the subchondral bone plate is perforated. 21 The phenomenon has also been seen in equine studies in which chondral defects were treated with microfracture and concentrated bone marrow aspirate. 22 In human patients, the quality of cartilage repair following microfracture is variable and inconsistent for unknown reasons and younger patients have better clinical outcomes and quality of cartilage repair than older patients. 23,24 Reasons for variation in clinical outcome after microfracture remain unclear but potentially may be explained by factors such as preexisting inflammation or genetic predisposition and there is limited evidence that microfracture should be accepted as gold standard for the treatment of cartilage lesions in the knee joint. 15 Nevertheless, the technically simple and inexpensive nature of this treatment makes microfracture a popular treatment for chondral and subchondral articular lesions in human and equine patients.

| Mosaicplasty
Arthroscopic mosaic arthroplasty or mosaicplasty is commonly used in human surgery to repair large chondral defects by harvesting osteochondral cores from nonweightbearing areas and transplanting these to the affected site. 25 This approach has been evaluated experimentally in the equine carpus and stifle. In one study three osteochondral grafts were harvested arthroscopically from the femoropatellar joint and transplanted to the third carpal bone. At 9 months post-operatively osteochondral grafts in the third carpal bone had less proteoglycans, leaving the cartilage softer and less resistant compared to surrounding cartilage.
Six of 18 grafts had histological evidence of cartilage degeneration and it was suggested that discrepancy in cartilage thickness between donor and recipient site was a major limitation with this technique. 26 In another study, osteochondral plugs were harvested from the cranial surface of the medial femoral trochlea and implanted into defects on the weightbearing surface of the contralateral medial femoral condyle in five horses. After 12 months, 50% of the grafts showed surgical procedure. A single step procedure has also been described, first in an equine model and then applied in human patients. 28,29 In the equine stifle ACI secured with a periosteal flap and fibrin glue led to an overall improvement of histological scores compared to nongrafted defects but the repair tissue was not different in composition from fibrocartilaginous repair and the study had a short follow-up period of only 8 weeks. 30 Combining the ACI procedure with growth factors (IGF-1) and using genetic overexpression of IGF-1 and BMP-7 stimulated early repair within the cartilage defect, but in the longterm results were less significant. 31 The same research group repeated the MACI experiment with longer duration (53 weeks) and a larger number of animals. 35 The biomechanical proprieties of the repair tissue in this study were presented in a separate publication. 36

| Mesenchymal stem cells and progenitor cells
The first experimental study using bone marrow aspirate for treatment of clinically relevant chondral defects in an equine model compared concentrated bone marrow aspirate concentrate (BMC) in combination with microfracture with microfracture alone. All outcome scores and magnetic resonance imaging supported improved healing in the bone marrow group but no biomechanical testing of the repair tissue was performed. 22 Due to concerns about potential undesirable subchondral bone changes after microfracture, the study was repeated with a slightly different protocol and with a longer follow-up ( A study using bone marrow-derived-MSCs (BMSC) in fibrin for repair of full thickness articular defects in the lateral trochlear ridge of the femur had promising results at 1 month, but did not show significant differences at 8 months. 39 In another study in the same model, BMSCs in a fibrin/platelet-rich plasma (PRP) hydrogel showed inferior repair compared to the fibrin/PRP injected controls. In 4 out of 12 cases the BMC-enriched fibrin/PRP defects was associated with bone formation within the defect. 40

| Allografts, autologous grafts and bioprinting
Artificial biological scaffolds can be manufactured in a more reproducible way than natural scaffolds and will also provoke less immune-  A decellularised matrix scaffold has been tested first in a shortand subsequently in a long-term equine model. A collagen-derived matrix was implanted alone or combined with a 3D-printed calcium phosphate cement-based scaffold to fill osteochondral defects in the middle trochlear ridge of the femur of eight adult horses (Figure 3).

| Biphasic grafts and zonal constructs
The hypothesis that the composite scaffold would lead to overall better anatomical reconstitution and that the chondral portion would heal with repair tissue closely resembling hyaline cartilage could not be confirmed. 44 After 6 months, histology and biochemistry showed predominantly fibrotic repair tissue, without significant differences between groups. The bony portion of the scaffold was, however, well integrated within the surrounding bone tissue (Figure 4).

| A matter of form or function
The high number of equine ongoing studies concerning chondral and osteochondral defect treatment and the diversity of approaches used, is proof of a still missing long-term solution. In fact, no technology has yet brought convincing evidence of efficacious long-term cartilage repair. The reason for the relative stagnation of progress in the field may not only be technical in nature, but may also relate to more conceptual issues, such as the use of terminology and the choice of animal models.
Positive results in the reported studies mostly refer to the presence of collagen type 2 or glycosaminoglycans (GAG) in the (immuno)histochemical analysis of the repair tissue ( Figure 5). Although the presence of these components is obviously necessary as a first step, the appreciation of their presence within repair tissue should perhaps be recon-

| Terminology
Reviewing the studies summarised above, it is noticeable that the

| Equine experimental models
In terms of translation of findings from experimental studies summarised above to clinical practice, it is critical to recognise that all joints involved in the cited studies were 'healthy' to begin with. This  Furthermore, these species lack one of the great advantages of the equine model, that is being not only an experimental animal but also a target animal with a clear and unmet clinical need.

| Future research
At this time, our opinion is that there are two major avenues that could produce substantial progress in the attempts to restore function in a joint with acute or chronic osteochondral defects and in preventing development of osteoarthritis.
The first would be to create novel strategies based on the developmental biology of articular cartilage, mimicking the embryonic and fetal mechanisms that produce the tissue to begin with. Unfortunately, this ambitious goal remains elusive at the moment, mainly because currently there is poor understanding of the developmental biology of articular cartilage. It is still unclear how articular cartilage formation initiates in the embryo and how it is brought to completion and maturity in young adult life. 63 Investing in more basic research would seem to be the most logic path to follow, although (at least in the short term) it is less economically appealing than seeking and finding a commercially usable product to fill osteochondral defects with.
The second way to improve therapeutic efficacy would be to focus attempts at cartilage repair more on functional results.
Proteoglycan renewal can take up to 25 years in a joint whereas the half-life of collagen is estimated to range from several decades to up to 400 years. 55 The current paradigm of tissue engineering aims to create the right conditions for the production of new natural tissue. 64 Perhaps, the focus should shift from the elusive goal of regeneration to achieving functional long-term repair. In this light, the resorbable nature of the implant materials might be questioned and materials with half-life that are similarly as long as those of the natural components of the extracellular matrix, for example polyurethane elastomers might be considered. 65 The need for clinically and commercially viable methods promotes 'simplification' of the biological process of osteochondral repair. Strategies to achieve this include eliminating the need for two procedures, by utilising 'press-fit' techniques with no additional need for fixation and by lowering regulatory hurdles by abstention of use of culture-expanded cell populations (Figure 8).
In conclusion, we believe future research should focus on function of repair cartilage rather than on visual appearances or biochemical analyses. Mere production of collagen type II and glycosaminoglycans is not enough to create functional properties similar to those of native cartilage, as function is determined by the inseparable combination of constituting elements with a highly specific architectural arrangement. 66 Future studies should always include assessment of repair cartilage structure and accurate biomechanical testing. In order to avoid disappointment when testing a successful short-term result in long-term studies, 31 The justified concerns on the expense and high expertise level needed for these procedures make it clear how the optimal solution to repair of chondral and osteochondral defects in the horse is yet to be found. Nevertheless, our view is that the short-term benefits (2-3 years) of microfracture may still be of interest in the equine racing industry given the inexpensive and simple nature of the treatment.

E TH I C A L A N I M A L R E S E A RCH
No ethical approval needed.

OWN E R I N FO R M E D CO N S E NT
Not applicable.

AUTH O R CO NTR I B UTI O N S
M. C. Fugazzola designed and prepared the manuscript. P. R. van Weeren contributed with intellectual input and editing. M. C. Fugazzola F I G U R E 8 Examples of a biphasic scaffold. The bony portion of the osteochondral defect is filled with the ceramic scaffold, whereas the hydrogel which is previously cast and cross-linked on top, fills the cartilage interface. These type of osteochondral plugs can be placed into created defects with a 'press-fit' application. In the left image: osteochondral defect on the medial ridge of the femur of a horse, filled with a biphasic scaffold (right image) had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Both authors have approved the final version of the manuscript.

CO N FLI C T O F I NTE R E S T
No competing interests have been declared.