Introduction

The menisci are C-shaped fibrocartilaginous structures that serve an important biomechanical function. Meniscal tears or degeneration, and total or partial meniscectomy has been linked to secondary osteoarthritis. 20 - 50% of middle aged and older patients without osteoarthritis have structural meniscus damage or degeneration (Englund M et al, Nat Rev Rheumatol. 2012). This incidence of meniscus pathology rises to 70-90% in knees with documented osteoarthritis.

Meniscectomy (total or partial) is the recommended procedure for symptomatic tears or degeneration of the meniscus. Tears in the vascular zone can be repaired in the anticipation that the vascularity will support healing. However, tears in the avascular zone have little to no capacity of repair. Despite the potential for secondary osteoarthritis even after meniscectomy, there is as yet no FDA-approved treatment for meniscal replacement. While allograft meniscal replacement is clinically permitted, clinical outcomes are generally poor.

There is therefore an unmet need for cell-based meniscus tissue engineering. There are obvious challenges in harvesting autologous cells from normal menisci. We studied the function of alternative sources of cells such as synoviocytes, mesenchymal stem cells, and cells from the infrapatellar fat pad. We have also explored potential for fabricating anisotropic meniscal tissue using electrospinning to mimic meniscal fiber architecture.

Content

We constructed layers of aligned polylactic acid (PLA) electrospun scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to form meniscus-like neotissues (Baek J et al, J Orthop Res, 2015). PLA electrospun scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. The morphology and mechanical properties of PLA scaffolds were influenced by fiber direction of the scaffolds. PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, and COMP. Overall, electrospun materials, which possess mechanical strength approaching that of native meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies.

We then investigated potential for electrospinning type 1 collagen seeded with human meniscus cell and implanted in meniscal defects (Baek J et al, Tissue Engineering, 2016). Surgical defects resembling ‘‘longitudinal tears’’ were created in the avascular zone of ex vivo bovine meniscus explants and implanted with cell-seeded collagen scaffolds. Ex vivo implantation with cell-seeded collagen scaffolds resulted in neotissue that was significantly better integrated with the native tissue than acellular collagen scaffolds or untreated defects on histology, immunohistochemistry, mechanical testing, and magnetic resonance imaging.

We then demonstrated proof-of-concept that electrospun collagen scaffolds support neotissue formation and that cells from the infrapatellar fat pad (IPFP) cells have potential for meniscus regeneration (Baek J et al, Tissue Engineering, 2018). We examined meniscus tissue generation from several human cell sources including meniscus cells derived from vascular and avascular regions, bone marrow-derived mesenchymal stem cells (hMSC), synovial cells, and IPFP. All cells were seeded onto multilayered electrospun collagen type I scaffolds. TGFβ1 and TGFβ3 treatment increased COL1A1, COMP, TNC, and SCX gene expression and deposition of collagen type I protein. Overall, IPFP cells generated meniscus-like tissues with higher meniscogenic gene expression, greater mechanical properties, and better cell distribution compared to other cell types studied.

Achieving appropriate cell density and distribution within densely tissue-engineered matrix is a challenge. Cells seeded on top of thick fibrous matrices often do not migrate into the deeper layers. To address this challenge, we developed a method of combined electrospraying of cells and electrospinning of fibers towards a one-step fabrication of live tissue (Baek J, et al, ORS 2017). Collagen type I was electrospun to generate aligned ES scaffolds. Human meniscal cells were simultaneously electrosprayed into the fibers, during the electrospinning. Our method of electrospraying cells enhanced cell attachment onto engineered collagen scaffolds, generated neo-meniscus tissue formation and maintained meniscus phenotype with high expression of COL1A1, ACAN, SOX9, and COMP. More importantly, simultaneously electrospraying cells and electrospinning significantly increased cell density and mechanical properties compared to superficial seeding of cells. We demonstrated that meniscal cells can be electrosprayed with low cytotoxicity, high meniscogenic gene expression, synthesis of collagen-rich matrix, and enhancement of mechanical properties. This approach of generating thick 3D cellular collagen nanofibrous scaffolds has potential in engineering a meniscus graft with the desired biological and mechanical qualities for meniscal replacement.

In conclusion, we conducted a series of studies culminating in electrospinning of nano fibers and electrospraying cells towards a novel approach to meniscus tissue engineering. These methods advance the fabrication of tissue matrices with optimal cell density and distribution to enhance engineering of 3D constructs for biological meniscal replacement.

References

Englund M et al, Nat Rev Rheumatol. 2012

Baek J et al, Tissue Engineering, 2016

Baek J et al, Tissue Engineering, 2018

Baek J, et al, ORS 2017

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.