The effects of staged intra-articular injection of cultured autologous mesenchymal stromal cells on the repair of damaged cartilage: a pilot study in caprine model

Chondral injuries often afflict the young owing to their athletic lifestyle and the high impact mechanical loading subjected to their joints during routine activities [1]. Injuries, especially those which result in focal cartilage defects, lead to an immediate loss in articular surface smoothness, resulting in the increase in tissue attrition. This triggers a series of changes to the subchondral bone which can lead to joint pain and dysfunction which, if left untreated, can lead to osteoarthritis [2]. Although in rare cases, it has been reported that the full-thickness chondral defect can heal spontaneously, the resultant repair tissue forms fibrous cartilage which will eventually lead to tissue degeneration [3]. This poses serious issues, as many of these patients would present with irreversible cartilage damage by the time they seek help from health care providers. The urgency to resolve this problem becomes more apparent when such conditions involve the young with many years of productivity still expected of them [4, 5].

To halt the progress of cartilage deterioration as the result of focal cartilage damage, several conventional treatments have been suggested, including marrow stimulation procedures [6]. This technique involves creating channels through the subchondral bone that allow access for blood and marrow elements to reach the damaged surface, which have been suggested to contain the essential ingredients by which cartilage healing occurs [7]. However, the superclot that forms in the defect as it matures with time tends to develop into a mixture of fibrocartilage and hyaline-like repair tissue. Nevertheless, using this technique, Steadman et al. were still able to demonstrate that bone marrow stimulation (BMS) can produce good outcomes in short- to mid-term follow-up of patients who had undergone this procedure [8]. This success, however, is short-lived and, thus, further improvements to this technique may lead to better long-term outcomes.

In recent years, it has been suggested that the use of intra-articular injection of mesenchymal stromal cells (MSCs) may result in moderately good repair outcomes, even in joints that have undergone mild degeneration [9]. In several studies, it has been shown that MSCs in circulating synovial fluid not only restores cartilage integrity but also halts the progression of cartilage degeneration [10]. In both BMS and MSC injection to the joint, there have been many suggestions to the mechanisms involved that have resulted in the positive outcomes observed; however, none have been sufficient to provide a satisfactory or conclusive explanation [11]. It may be the case that the combination of both techniques may result in a synergistic outcome, thus providing superior tissue repair. Based on our extensive literature search, there have not been any published results that support this notion, although one study using mononuclear cells and not MSCs suggests that this may be the case [12]. To prove whether such a synergistic effect exists, a study was conducted using a focal cartilage defect in a caprine model treated with BMS with or without intra-articular injection of MSCs. Outcome measures were conducted six months post-operative using macroscopic, histology, and selective cartilage protein and gene expression assessments.

In the present study, the potential of autologous BM-MSCs was compared to a standard treatment for focal cartilage defect, that is, subchondral drilling as the method for marrow stimulation, in caprine models. Data from the present study suggest that the use of BM-MSCs as an adjunct therapy provides certain levels of improvements to this method of cartilage repair.

Although the authors of the present paper are only aware that the present study conducted is the first to demonstrate such findings, there were several previous studies using almost similar techniques that are worth comparing. In 2009, Saw et al.[12] described the use of bone marrow aspirates and hyaluronic acid (HA) in improving the repair of BMS-treated chondral defects. In their experimental study, they found that from using this technique the cartilage repair produced integrated hyaline-like tissue. This led to their claim that there may be a redundancy of practicing MSCs’ isolation and in vitro expansion, as marrow aspirates may be sufficient to achieve good results. However, it is noteworthy that the limitations in this study were obvious. Among these was the fact that the conclusion was based on a single histological score using four goats in each group. It was statistically weak and lacked sufficient measured parameters. The authors then published a cohort study involving 50 patients but this time using peripheral derived stem cells [17]. They claim that the outcome was good, based on limited representative histological scoring and MRI only. However, there are several issues that this paper did not address, and this could have led to the over-rated findings of that reported study. While the authors mentioned that the cells obtained from the peripheral blood were stem cells, there was no evidence to support such claims since there was no cellular characterization performed. Furthermore, the cells obtained do not fulfill any criteria that identify them as stem cells, that is, the ability for higher cell potency, such as multipotency. With no cell sorting or selection performed, at best the cells used can be described as mixed mononuclear cells. There also appears to be a discrepancy between the histological and patient outcome scores which does not appear to correlate, demonstrating that the outcome may not have been as good as what has been concluded. The issue in this study, and that of others, would have been whether histological scores reflect the good outcomes that are measured following any tissue repair. In many of the studies we reviewed, although histological analyses were used, other parameters were also measured which include patient scores, protein and/or gene expression, and functional biomechanical analyses [18, 19]. In these studies, correlations between histological scores and the final outcomes were always present. This is similar to what was found in our study. There was strong correlation found between these scores and that each of the measured parameter appears to corroborate the findings of the other parameters. We can, therefore, safely assume that the reports from our study are more likely to reflect better accuracy and thus are more reliable as compared to the study reporting the use of marrow or peripheral blood mononuclear cells.

In a separate study by Lee et al., chondral defects treated using bone marrow MSCs only, contributed to superior cartilage repair [20]. This seems to support our findings that when a combination of MSCs and BMS is used, a superior outcome can be expected. Unlike the previous study by Saw et al., which lacked characterization to claim that the cells were stem cells, the study by Lee et al. demonstrated several features which suggest that the cells used were as such, albeit limited in terms of today’s standard since no surface CD marker expressions were investigated in their study. One advantage their study offered over the presently reported article is that observations were carried out in two different time points, which showed progressive improvements. In contrast, ours, which lasted up to 29 weeks, merely showed good repair outcomes in the treated group. One may argue that given a larger time scale, those goats not supplemented may have had comparable outcomes. Nevertheless, if no improvements were observed at six months, we can safely say that a treatment has most likely reached its ultimate endpoint of progression.

There were several studies that used intra-articular injection of stem cells; however, these involved smaller animals without marrow stimulations and are usually osteoarthritic models as opposed to focal cartilage defects. It is worth noting that osteoarthritis represents a degenerative stage which is usually the end stage of a diseased condition, while focal cartilage defects represent the pre-degenerative state at best. Among the studies worth mentioning is that by Agung et al. [21], which evaluated the use of MSCs in rodents. It was demonstrated that using MSCs injected into injured knee joints could mobilize these cells to the injured area, contributing to tissue regeneration. Similarly, this was observed by our co-researchers [10]. They examined the effects of MSCs, HA and the combination of HA-MSCs in treating osteoarthritis (OA) in a rat model. Their study suggested that the use of either HA or MSCs effectively reduces OA progression better than their combined use, which suggests that MSCs have modulatory effects on damaged articular cartilage. These studies appear to suggest that the findings of our study are not unexpected as MSCs produce positive healing effects when injected intra-articularly.

The reason for the fibrous tissue formation instead of hyaline cartilage seen in the BMS only group may be explained by the inflammatory response invoked due to trauma. It is suggested that the combination of BMS and BM-MSC may have slowed this process, thereby promoting hyaline cartilage regeneration as opposed to fibrocartilage formation. The reason for this, and even for those observed in our study, is rather contentious, since there are already many scientists debating the issue as to whether the improvement seen in such cases is the direct result of the repair by the MSCs, or whether it is the factors produced by MSCs, or even perhaps the proteins they attract which ultimately result in direct repair or retardation of the inflammatory process [11]. In previous reports, MSCs are said to be able to secrete a broad spectrum of bioactive molecules that have immunoregulatory [22‐24] and/or regenerative activities [25]. Bioactive factors secreted by MSCs have been shown to inhibit tissue scarring; suppress apoptosis, inflammatory and fibrotic; stimulate angiogenesis where MSCs secrete a growth factor, VEGF (Vascular Endothelium Growth Factor), which stimulates vascularization; and enhance mitosis of tissue-intrinsic stem or progenitor cells. Other studies have shown that MSCs attach to the defect sites while others have shown that in the transplanted site MSCs regress dramatically to a point that they are no longer viable [26, 27]. Regardless of the mechanisms involved, the present study clearly demonstrated a correlation between the use of MSCs with superior tissue repair, which will require further investigations if we wish to elicit the mechanisms involved. It must be stressed that the data presented here were not only based on macroscopic and histological assessments, but also include other investigations, such as GAG content and selective cartilage gene expression, that is, SOX9 and Collagen II, which demonstrated a significant repair process as compared to BMS alone. This demonstrates the complex relationship in the healing processes involved, which justifies our claims that the use of MSCs as an intra-articular adjuvant to BMS improves the many facets of articular joint healing and that the observation made is not merely by chance.

Despite the robust findings presented here, our study presents several limitations that are worth noting. Financial restriction prohibited functional assessment of the repaired site, such as biomechanical testing, to be conducted. Thus, it was difficult to ensure whether our observation of the histologic appearance of the regenerated cartilage is directly proportional to the biomechanical function of the tissues. Nevertheless, we consider that this study still provides valuable findings since the conclusions were drawn using well-accepted experimental techniques utilized in several other previous studies [28, 29]. Introduction of another treatment arm that utilizes a standard treatment approach for cartilage repair, such as hyaluronic acid [30] injection, although not mandatory, could have led to a more comprehensive conclusion. Improvement could also be made to the study by using a larger number of animals, thereby increasing the significance levels in this study. Nevertheless, one could argue that statistically this number of samples was deemed appropriate as is. It is well known that repair of articular cartilage lesions remodels with time [6, 31‐33]. Evaluation of the result at only one time point, at six months, is hence unable to detect progressive remodeling that has been shown to occur beyond a year [6]. This should be addressed in future studies.

In conclusion, our preliminary study demonstrates that within reasonable limits, the use of supplementary intra-articular injection of BM-MSCs produced modulatory effects on the repair produced by marrow stimulation. This study, however, will require further evaluations and more convincing results using more rigorous experiments. These questions will be answered in our ongoing study, which is presently being conducted based on the results of the currently reported pilot project.