Platelet rich concentrate enhances mesenchymal stem cells capacity to repair focal cartilage injury in rabbits

Abstract

Background

It has been previously suggested that the use of regenerative promoters, which include bone marrow-derived mesenchymal stem cells (MSCs) or natural growth factors supplement such as platelet-rich concentrate (PRC) could promote cartilage regeneration. However, the notion that the concurrent use of both promoters may provide a synergistic effect that improves the repair outcome of focal cartilage injury has not been previously demonstrated. This study was thus conducted to determine whether the concomitant use of PRC could further enhance the reparative potential of MSCs encapsulated in alginate transplanted into focal cartilage injury in rabbits.

Methods

Artifically created full thickness cartilage defects were made on the weight-bearing region of medial femoral condyles in bilateral knees of New Zealand White rabbits (N = 30). After one month, the right knee was treated with either i) PRC (n = 10), ii) MSCs (n = 10), or, iii) a combination of PRC and MSCs (PRC + MSC) (n = 10), all encapsulated in alginate. The left knee remained untreated (control). Rabbits were sacrificed at 3 and 6 months after treatment. Cartilage tissue regeneration was accessed using ICRS morphologic scoring, histologic grading by O'Driscoll scoring, immunohistochemical staining and quantitative analysis of glycosaminoglycans (GAG) per total protein content.

Results

At 3 months, transplantation using PRC alone was equally effective as MSCs in inducing the repair of cartilage defects. However, PRC + MSC resulted in significantly higher ICRS and O’Driscoll scores (p < 0.05) as compared to other groups. The regenerated tissues from the PRC + MSC group also had stronger staining for Safranin-O and collagen type II. By 6 months, in addition to superior ICRS and O’Driscoll scores as well as stronger staining, glycosaminoglycan per total protein content was also significantly higher (p < 0.05) in the PRC + MSC group (3.4 ± 0.3 μg/mg) as compared to the MSC (2.6 ± 0.2 μg/mg) or PRC (2.1 ± 0.2 μg/mg) groups.

Conclusion

PRC enhances the reparative effects of MSC in treating focal articular cartilage injuries.

Introduction

Focal injury or defect of articular cartilage is a prevalent orthopaedic problem, particularly amongst individuals participating in high impact sports activities [1]. If left untreated, the initial chondral or osteochondral lesion may progress further, predisposing the individual to early onset of osteoarthritis. Successful treatment of articular cartilage defect in the knee has continued to be a formidable challenge for clinicians, owing to the well-known poor intrinsic healing capacity of normal cartilage tissue. Nevertheless, as the knowledge of cartilage biology expands, many novel potential treatment strategies for cartilage repair have been increasingly explored in the recent years. This includes the use of regenerative promoters such as mesenchymal stem cells (MSCs) and growth factors. MSCs have been shown to promote cartilage regeneration through the production of cartilage matrix, whilst several growth factors have been shown to stimulate cartilage resident cells i.e. chondrocytes to produce repaired tissues [2]. The ability of MSCs to undergo self-renewal and multi-lineage differentiation, as well as having immunomodulatory and anti-inflammatory properties, renders them as an attractive potential therapeutic agent for tissue repair [3]. Accordingly, many approaches have been developed to augment the reparative potential of MSCs. One such effort is through the use of naturally occurring source of growth factors such as platelet-rich concentrate (PRC). PRC in essence, is a concentrated suspension of platelets above baseline levels. In many instances, PRC may be prepared from the re-suspension of platelets in liquid suspensions such as normal saline. PRC contains various mitogenic and angiogenic growth factors such as platelet-derived growth factor (PDGF) − AA, −BB, −AB, transforming growth factor β1 (TGFβ −1) and −2, insulin growth factor-1 (IGF-1), and fibroblast growth factor (FGF), which have been shown to stimulate cell proliferation, cellular protein expression, and tissue regeneration [4]. Supplementing platelet-rich plasma (PRP) to culture medium has been shown to enhance chondrocyte proliferation and matrix synthesis [5] as well as improve chondrogenic differentiation of MSCs in vitro [6]. In animal studies, PRP has been shown to enhance articular cartilage repair when used in conjunction with microfracture for the repair of chondral defects [[7], [8], [9], [10]], or when used for treatment of osteochondral defects [[11], [12], [13], [14], [15]]. Direct PRP injections administered after subchondral bone drilling for the repair of chondral defects have also been shown to produce excellent functional outcome in clinical studies [[16], [17], [18]]. In all instances, the therapeutic potential of platelet-rich preparation in vivo has been demonstrated only when it was used in conjunction with some form of surgical intervention, such as following bone marrow-stimulation procedure. Considering the fact that microfracture or subchondral bone drilling would itself stimulate cells from the bone marrow to migrate and initiate in situ cartilage repair, it remains unclear to what extent platelet-rich preparation on its own is sufficiently efficacious in promoting healing of cartilage injury in vivo without the influence of other potential confounding factors. More importantly, it is also unclear whether the use of platelet-rich concentrate could further enhance the capacity of MSCs to repair focal cartilage injury in vivo since the synergistic effect of both factors have not been previously demonstrated in an animal model of this injury. Therefore, the aim of this study was to determine the effectiveness of platelet-rich concentrate (PRC) encapsulated in alginate beads in enhancing articular cartilage repair in an in vivo rabbit model of focal cartilage injury, and to investigate whether implantation of PRC together with MSCs encapsulated in alginate into the defect site could further enhance repair of the injury, as reflected by gross morphology, histology, and glycosaminoglycan levels of the regenerated tissue. We hypothesize that treatment with a combination of PRC and MSCs encapsulated in alginate will enhance the quality of repair of the full thickness chondral defects as compared to PRC and MSC in alginate alone.