Purpose

Treatment of osteoarthritis with novel injectable therapies could be improved by targetting the lesion sites in the articular cartilage. We have previously shown that degradation of Type II collagen in osteoarthritis occurs at the surface of the articular cartilage, leading to an accumulation of Type II collagen gelatin (1), a prime candidate for therapeutic targetting. Gelatinases A and B contain a catalytic domain as well as three fibronectin-like modules that form the collagen binding domain (CBD). The purpose of this study was to determine which of these modules is most important for binding to Type II collagen gelatin and to enhance the binding to this gelatin through mutations in its structure. The long-term goal is to deliver either drugs or cells (see Ref. 2) directly into the lesion sites.

Methods and Materials

Full length CBD, individual modules of CBD or mutations of these modules were expressed in E.coli shuffle cells, purified and then biotinylated before being tested for their ability to bind to denatured collagen types I and II by direct binding ELISA.

Results

CBD was found to have 10-fold higher binding (ie 10-fold lower Kd) to Type I collagen gelatin than Type II (Figure 1). Module 2 bound more tightly to gelatin than modules 1 or 3 (not shown), therefore we constructed a mutant CBD using three repeats of module 2 (222) and tested its binding . 222 had a 14-fold higher affinity for Type II collagen gelatin, with a Kd that was similar to that of is binding to Type I collagen gelatin (Figure 2).

Conclusion

The CBD mutant, protein 222, has a high binding capacity for Type II collagen gelatin and so could be used to target cells and drugs into cartilage lesions.

References

1. A. P. Hollanderet al., Journal of Clinical Investigation 96, 2859-2869 (1995)

2. J. P. Armstronget al., Nat Commun 6, 7405 (2015)

Introduction

Meniscal tears are a common sporting injury and most of them occur in the white (avascular) zone, where attempts at repair using conventional techniques usually fail. There is strong evidence that conservative treatment by partial meniscectomy increases the risk of the development of osteoarthritis (OA) within a few years of surgery (1). Mesenchymal stromal/stem cells (MSCs) could potentially be used to drive meniscal repair (2) however a delivery method is needed that enables repair of complex tears in adult humans with a low intrinsic capacity for tissue regeneration.

MSCs were originally considered to function as typical stem cells, with the capacity to differentiate to chondrocytes and other mesenchymal lineages and thereby participate in repair process. However there has been a growing view that these cells have another, perhaps more important function, namely to drive endogenous repair processes through interaction with cells in the target tissues where they are implanted (3). We have exploited this trophic capacity for MSCs in developing a strategy for meniscal repair.

Content

We have spent the past decade exploring the biology of MSCs and understanding how best to deliver them into meniscal tears in order to restore the injured tissue to its natural state and avoid meniscectomy (4-6). In our initial work (4) we used an articular cartilage repair model to study how to integrate two pieces of cartilage in vitro and developed the concept of a "Cell Bandage" comprising cells (initially we used chondrocytes) and a carefully selected scaffold. The Cell Bandage was inserted between the pieces of cartilage and delivered the cells across the interface. We went on to explore the Cell Bandage concept in meniscal repair in vitro and moved to the use of undifferentiated MSCs and a collagen scaffold (5). We tested Cell Bandage in a sheep model of meniscal injury and finally undertook a first in human study using autologous MSCs in 5 patients with encouraging results (6). In future we intend to develop an allogeneic Cell Bandage that can be made readily available to larger numbers of patients.

References

1. S. G. Muthuri, D. F. McWilliams, M. Doherty, W. Zhang, History of knee injuries and knee osteoarthritis: a meta-analysis of observational studies. Osteoarthritis Cartilage19, 1286-1293 (2011).

2. J. M. Murphy, D. J. Fink, E. B. Hunziker, F. P. Barry, Stem cell therapy in a caprine model of osteoarthritis. Arthritis and rheumatism48, 3464-3474 (2003).

3. A. I. Caplan, D. Correa, The MSC: an injury drugstore. Cell Stem Cell 9, 11-15 (2011).

4. M. B. Pabbruweet al., Repair of meniscal cartilage white zone tears using a stem cell/collagen-scaffold implant. Biomaterials 31, 2583-2591 (2010).

5. M. B. Pabbruwe, E. Esfandiari, W. Kafienah, J. F. Tarlton, A. P. Hollander, Induction of cartilage integration by a chondrocyte/collagen-scaffold implant. Biomaterials 30, 4277-4286 (2009).

6. M. R. Whitehouseet al., Repair of Torn Avascular Meniscal Cartilage Using Undifferentiated Autologous Mesenchymal Stem Cells: From In Vitro Optimization to a First-in-Human Study. Stem Cells Transl Med 6, 1237-1248 (2017).

Acknowledgments

We are grateful to The Wellcome Trust and Innovate UK for funding some of the work reported here.