Brian C. Pridgen

Flexor Tendon Tissue Engineering: Acellularization of Human Flexor Tendons with Preservation of Biomechanical Properties and Biocompatibility

OBJECTIVE Acellular human tendons are a candidate scaffold for tissue engineering flexor tendons of the hand. This study compared acellularization methods and their compatibility with allogeneic human cells. METHOD Human flexor tendons were pretreated with 0.1% ethylenediaminetetracetic acid (EDTA) for 4  h followed by 24  h treatments of 1% Triton X-100, 1% tri(n-butyl)phosphate, or 0.1% or 1% sodium dodecyl sulfate (SDS) in 0.1% EDTA. Outcomes were assessed histologically by hematoxylin and eosin and SYTO green fluorescent nucleic acid stains and biochemically by a QIAGEN DNeasy kit, Sircol collagen assay, and 1,9 dimethylmethylene blue glycosaminoglycan assay. Mechanical data were collected using a Materials Testing System to pull to failure tendons acellularized with 0.1% SDS. Acellularized tendons were re-seeded in a suspension of human dermal fibroblasts. Attachment of viable cells to acellularized tendon was assessed biochemically by a cell viability assay and histologically by a live/dead stain. Data are reported as mean±standard deviation. RESULT Compared with the DNA content of fresh tendons (551±212  ng DNA/mg tendon), only SDS treatments significantly decreased DNA content (1% SDS [202.8±37.4  ng DNA/mg dry weight tendon]; 0.1% SDS [189±104  ng DNA/mg tendon]). These findings were confirmed by histology. There was no decrease in glycosaminoglycans or collagen following acellularization with SDS. There was no difference in the ultimate tensile stress (55.3±19.2 [fresh] vs. 51.5±6.9 [0.1% SDS] MPa). Re-seeded tendons demonstrated attachment of viable cells to the tendon surface using a viability assay and histology. CONCLUSION Human flexor tendons were acellularized with 0.1% SDS in 0.1% EDTA for 24  h with preservation of mechanical properties. Preservation of collagen and glycoaminoglycans and re-seeding with human cells suggest that this scaffold is biocompatible. This will provide a promising scaffold for future human flexor tendon tissue engineering studies to further assess biocompatibility through cell proliferation and in vivo studies.

Optimization of Human Tendon Tissue Engineering: Peracetic Acid Oxidation for Enhanced Reseeding of Acellularized Intrasynovial Tendon

Background: Tissue engineering of human flexor tendons combines tendon scaffolds with recipient cells to create complete cell-tendon constructs. Allogenic acellularized human flexor tendon has been shown to be a useful natural scaffold. However, there is difficulty repopulating acellularized tendon with recipient cells, as cell penetration is restricted by a tightly woven tendon matrix. The authors evaluated peracetic acid treatment in optimizing intratendinous cell penetration. Methods: Cadaveric human flexor tendons were harvested, acellularized, and divided into experimental groups. These groups were treated with peracetic acid in varying concentrations (2%, 5%, and 10%) and for varying time periods (4 and 20 hours) to determine the optimal treatment protocol. Experimental tendons were analyzed for differences in tendon microarchitecture. Additional specimens were reseeded by incubation in a fibroblast cell suspension at 1 × 106 cells/ml. This group was then analyzed for reseeding efficacy. A final group underwent biomechanical studies for strength. Results: The optimal treatment protocol comprising peracetic acid at 5% concentration for 4 hours produced increased scaffold porosity, improving cell penetration and migration. Treated scaffolds did not show reduced collagen or glycosaminoglycan content compared with controls (p = 0.37 and p = 0.65, respectively). Treated scaffolds were cytotoxic to neither attached cells nor the surrounding cell suspension. Treated scaffolds also did not show inferior ultimate tensile stress or elastic modulus compared with controls (p = 0.26 and p = 0.28, respectively). Conclusions: Peracetic acid treatment of acellularized tendon scaffolds increases matrix porosity, leading to greater reseeding. It may prove to be an important step in tissue engineering of human flexor tendon using natural scaffolds.

Overcoming the learning curve: a curriculum-based model for teaching zone II flexor tendon repairs.

Background: Repairs of zone II flexor tendons have benefited in recent years from modifications involving suture technique and configuration. These advances, however, present new obstacles in resident training. A focused tutorial incorporating a practical, hands-on exercise and standardization of technique may offer an effective low-risk, low-cost strategy for overcoming these challenges. Methods: Plastic surgery residents (n = 14) were asked to perform their preferred zone II flexor tendon repair using a tabletop exercise before and after a focused tutorial. The tutorial reviewed primary literature and presented a standardized technique. Repairs were photographed, tested for load strength, and analyzed to determine effectiveness of this teaching approach. Participants were retested at 6 months to evaluate for persistence of findings. Results: Posttutorial repairs required higher loads to generate a 2-mm gap (p < 0.001) and ultimate breakage (p < 0.001). Tendon purchase and resident confidence increased significantly. Subgroup analysis demonstrated significant improvements for both junior and senior residents. Retesting at 6 months revealed that gains were maintained over time. Conclusions: The authors created a practical educational model to teach zone II flexor tendon repair outside of the time- and error-sensitive confines of the operating room. Analysis of resident pretutorial repairs revealed common misconceptions in suture technique, strand count, and purchase. This may in part be attributable to the multitude of suggested repair techniques, difficulty in comparing data across multiple studies, and steep learning curve. Training programs can use this hands-on teaching exercise as part of a hand surgery simulation curriculum.

Syndactyly: Apert Syndrome

Apert syndrome is a rare congenital disorder characterized by craniosynostosis, midface hypoplasia, and bilateral syndactyly of the hands and feet. Characteristic hand anomalies include a short thumb with radial clinodactyly; involvement of the first web space with varying degrees of syndactyly between the thumb and index finger; complex syndactyly between the index, long, and ring fingers typically at the level of the distal interphalangeal joints or beyond; and variable degrees of syndactyly between the ring and small fingers. Reconstruction of the hand involves a series of staged procedures that are performed with the goal of minimizing the number of procedures, maximizing the functional outcome of the hand, and providing a favorable cosmetic result. The techniques and preferences of several groups and their outcomes are reviewed in this chapter.

99A: HUMAN FLEXOR TENDON TISSUE ENGINEERING: PRESERVATION OF BIOMECHANICAL STRENGTH IN ACELLULARIZED TENDONS

Methods: Human FDS tendons were treated with 0.1% EDTA for four hours. They were then treated for 24 hours with one of five detergent solutions in 0.1% EDTA: 1% Triton X-100, 1% TnBP, 0.1% TnBP, 1% SDS, or 0.1% SDS. An additional group was treated with 0.1% SDS for 24 hours but without EDTA pre-treatment. Outcomes were assessed histologically by H&E and SYTO Green nucleic acid stains and biochemically by DNA, glycosaminoglycan, and collagen assays. Biomechanical data were collected using FDP tendons acellularized with 0.1% SDS.

Overcoming the Learning Curve: A Novel Approach to Teaching Zone II Flexor Tendon Repairs

INTRODUCTION: Repair of Zone II flexor tendon injuries is a complex technique that has benefited from many advances in recent years. These advances present new challenges, however, for teaching within the operating room. (1-4) A focused tutorial incorporating a practical, hands-on model and standardization of repair technique may offer an effective low-risk, low-cost method for training future surgical residents. (5)