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A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

University College London

Northwick Park Hospital

The traumatic rupture of tendons is a common clinical problem. Tendon repair is surgically challenging because the tendon often retracts, resulting in a gap between the torn end and its bony insertion. Tendon grafts are currently used to fill this deficit but are associated with potential complications relating to donor site morbidity and graft necrosis. We have developed a highly reproducible, rapid process technique to manufacture compressed cell-seeded type I collagen constructs to replace tendon grafts. However, the material properties of the engineered constructs are currently unsuitable to withstand complete load bearing in vivo. A modified suture technique has been developed to withstand physiological loading and off load the artificial construct while integration occurs. Lapine tendons were used ex vivo to test the strength of different suture techniques with different sizes of Prolene sutures and tissue-engineered collagen constructs in situ. The data were compared to standard modified Kessler suture using a standard tendon graft. Mechanical testing was carried out and a finite element analysis stress distribution model constructed using COMSOL 3.5 software. The break point for modified suture technique with a tissue-engineered scaffold was significantly higher (50.62 N) compared to a standard modified Kessler suture (12.49 N, p<0.05). Distributing suture tension further proximally and distally from the tendon ends increased the mechanical strength of the repairs. We now have ex vivo proof of concept that this suture technique is suitable for testing in vivo, and this will be the next stage of our research. 

Development of a surgically optimized graft insertion suture technique to accommodate a tissue-engineered tendon in vivo.

Tissue-engineered collagen grafts to treat large tendon defects.

Tendon disease and injuries carry significant morbidity worldwide in both athletic and non-athletic populations. It is estimated that tendon injuries account for 30%-50% of all musculoskeletal injuries globally. Current treatments have been inadequate in providing an accelerated process of repair resulting in high relapse rates. Modern concepts in tissue engineering and regenerative medicine have led to increasing interest in the application of cell therapy for the treatment of tendon disease. This review will explore the use of cell therapy, by bringing together up-to-date evidence from in vivo human and animal studies, and discuss the issues surrounding the safety and efficacy of its use in the treatment of tendon disease. 

A review on the use of cell therapy in the treatment of tendon disease and injuries.

Biomaterials for tissue engineering include natural and synthetic polymers, but their clinical application is still limited due to various disadvantages associated with the use of these polymers. This uncertainty of the polymeric approach in tissue engineering launches an opportunity to address a key question: can we eliminate the disadvantages of both natural and synthetic polymers by combining them to form a synergistic relationship? To answer this question, we fabricated scaffolds from elastin, collagen, fibrin, and electrospun polycaprolactone (PCL) with different ratios. The material characterization of these scaffolds investigated degradation, water contact angle, angiogenesis by an ex ovo chorion allantoic membrane (CAM) assay, and mechanical and structural properties. Biological activity and specific differentiation pathways (MSC, adipogenic, osteogenic, myogenic, and chondrogenic) were studied by using human adipose-derived stem cells. Results indicated that all composite polymers degraded at a different rate, thus affecting their mechanical integrity. Cell-based assays demonstrated continual proliferative and viable properties of the cells on all seeded scaffolds with the particular initiation of a differentiation pathway among which the PCL/collagen/fibrin composite was the most angiogenic material with maximum vasculature. We were able to tailor the physical and biological properties of PCL-based composites to form a synergistic relationship for various tissue regeneration applications.

A Synergistic Relationship between Polycaprolactone and Natural Polymers Enhances the Physical Properties and Biological Activity of Scaffolds.

The use of decellularised matrices as scaffolds offers the advantage of great similarity with the tissue to be replaced. Moreover, decellularised tissues and organs can be repopulated with the patient's own cells to produce bespoke therapies. Great progress has been made in research and development of decellularised scaffolds, and more recently, these materials are being used in exciting new areas like hydrogels and bioinks. However, much effort is still needed towards preserving the original extracellular matrix composition, especially its minor components, assessing its functionality and scaling up for large tissues and organs. Emphasis should also be placed on developing new decellularisation methods and establishing minimal criteria for assessing the success of the decellularisation process. The aim of this review is to critically review the existing literature on decellularised scaffolds, especially on the preparation of these matrices, and point out areas for improvement, finishing with alternative uses of decellularised scaffolds other than tissue and organ reconstruction. Such uses include three-dimensional ex vivo platforms for idiopathic diseases and cancer modelling.

Decellularised scaffolds: just a framework? Current knowledge and future directions.

Reconstruction of a tendon rupture is surgically challenging as each end of the tendon retracts, leaving a substantial gap and direct repair is often not feasible. A tendon graft is required to bridge this defect and restore function. Presently, these gaps are filled with auto-, allo-, or synthetic grafts, but they all have clinical limitations. To address this issue, we developed tissue-engineered grafts by a rapid process using compressed type I collagen, which is the most dominant protein in the tendon. However, biomechanical properties were found to be unsuitable to withstand complete load-bearing in vivo. Hence, a modified suture technique was previously developed to reduce the load on the engineered collagen graft to aid integration in vivo. Using this technique, we tested engineered collagen grafts in vivo on a lapine model in three groups up to 12 weeks without immobilization. Gross observation at 3 and 12 weeks showed the bridge integrated without adhesions with a significant increase in the mechanical, structural and histological properties as compared to 1 week. Insertion of a tissue-engineered collagen graft using a novel load-bearing suture technique which partially loads in vivo showed integration, greater mechanical strength and no adhesion formation in the time period tested. This collagen graft has inherent advantages as compared to the present-day tendon grafts.

Engineering of a Functional Tendon Using Collagen As a Natural Polymer.

Due to the limitations of bone autografts, we aimed to develop new composite biomaterials with pro-angiogenic and osteogenic properties to be used as scaffolds in bone tissue engineering applications. We used a porous, cross-linked and slowly biodegradable fibrin/alginate scaffold originally developed in our laboratory for wound healing, throughout which deposits of calcium phosphate (CaP) were evenly incorporated using an established biomimetic method. Material characterisation revealed the porous nature and confirmed the deposition of CaP precursor phases throughout the scaffolds. MC3T3-E1 cells adhered to the scaffolds, proliferated, migrated and differentiated down the osteogenic pathway during the culture period. Chick chorioallantoic membrane (CAM) assay results showed that the scaffolds were pro-angiogenic and biocompatible. The work presented here gave useful insights into the potential of these pro-angiogenic and osteogenic scaffolds for bone tissue engineering and merits further research in a pre-clinical model prior to its clinical translation.

Pro-angiogenic and osteogenic composite scaffolds of fibrin, alginate and calcium phosphate for bone tissue engineering.

Current gold standard to treat soft tissue injuries caused by trauma and pathological condition are autografts and off the shelf fillers, but they have inherent weaknesses like donor site morbidity, immuno-compatibility and graft failure. To overcome these limitations, tissue-engineered polymers are seeded with stem cells to improve the potential to restore tissue function. However, their interaction with native tissue is poorly understood so far. To study these interactions and improve outcomes, we have fabricated scaffolds from natural polymers (collagen, fibrin and elastin) by custom-designed processes and their material properties such as surface morphology, swelling, wettability and chemical cross-linking ability were characterised. By using 3D scaffolds, we comprehensive assessed survival, proliferation and phenotype of adipose-derived stem cells in vitro. In vivo, scaffolds were seeded with adipose-derived stem cells and implanted in a rodent model, with X-ray microtomography, histology and immunohistochemistry as read-outs. Collagen-based materials showed higher cell adhesion and proliferation in vitro as well as higher adipogenic properties in vivo. In contrast, fibrin demonstrated poor cellular and adipogenesis properties but higher angiogenesis. Elastin formed the most porous scaffold, with cells displaying a non-aggregated morphology in vitro while in vivo elastin was the most degraded scaffold. These findings of how polymers present in the natural polymers mimicking ECM and seeded with stem cells affect adipogenesis in vitro and in vivo can open avenues to design 3D grafts for soft tissue repair.

Three dimensional porous scaffolds derived from collagen, elastin and fibrin proteins orchestrate adipose tissue regeneration.

Control of cell-surface interaction is necessary for biomaterial applications such as cell sheets, intelligent cell culture surfaces, or functional coatings. In this paper, we propose the emergent property of cell morphology as a design parameter in the bioengineering of cell-biomaterial surface interactions. Cell morphology measured through various parameters can indicate ideal candidates for these various applications thus reducing the time taken for the screening and development process. The hypothesis of this study is that there is an optimal cell morphology range for enhanced cell proliferation and migration on the surface of biomaterials. To test the hypothesis, primary porcine dermal fibroblasts (PDF, 3 biological replicates) were cultured on ten different surfaces comprising components of the natural extracellular matrix of tissues. Results suggested an optimal morphology with a cell aspect ratio (CAR) between 0.2 and 0.4 for both increased cell proliferation and migration. If the CAR was below 0.2 (very elongated cell), cell proliferation was increased whilst migration was reduced. A CAR of 0.4+ (rounded cell) favoured cell migration over proliferation. The screening process, when it comes to biomaterials is a long, repetitive, arduous but necessary event. This study highlights the beneficial use of testing the cell morphology on prospective prototypes, eliminating those that do not support an optimal cell shape. We believe that the research presented in this paper is important as we can help address this screening inefficiency through the use of the emergent property of cell morphology. Future work involves automating CAR quantification for high throughput screening of prototypes.

Prasad Sawadkar

Division of Surgery and Interventional Science

University College London

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects