Ioannis K. Angelidis

Tissue Engineering of Flexor Tendons: The Effect of a Tissue Bioreactor on Adipoderived Stem Cell-Seeded and Fibroblast-Seeded Tendon Constructs

PURPOSE Tissue-engineered flexor tendons could eventually be used for reconstruction of large tendon defects. The goal of this project was to examine the effect of a tissue bioreactor on the biomechanical properties of tendon constructs seeded with adipoderived stem cells (ASCs) and fibroblasts (Fs). METHODS Rabbit rear paw flexor tendons were acellularized and seeded with ASCs or Fs. A custom bioreactor applied a cyclic mechanical load of 1.25 N at 1 cycle/minute for 5 days onto the tendon constructs. Three additional groups were used as controls: fresh tendons and tendons reseeded with either ASCs or Fs that were not exposed to the bioreactor treatment and were left in stationary incubation for 5 days. We compared the ultimate tensile stress (UTS) and elastic modulus (EM) of bioreactor-treated tendons with the unloaded control tendons and fresh tendons. Comparison across groups was assessed using one-way analysis of variance with the significance level set at p<.05. Pairwise comparison between the samples was determined by using the Tukey test. RESULTS The UTS and EM values of bioreactor-treated tendons that were exposed to cyclic load were significantly higher than those of unloaded control tendons. Acellularized tendon constructs that were reseeded with ASCs and exposed to a cyclic load had a UTS of 66.76 MPa and an EM of 906.68 MPa; their unloaded equivalents had a UTS of 47.90 MPa and an EM of 715.57 MPa. Similar trends were found in the fibroblast-seeded tendon constructs that were exposed to the bioreactor treatment. The bioreactor-treated tendons approached the UTS and EM values of fresh tendons. Histologically, we found that cells reoriented themselves parallel to the direction of strain in response to cyclic strain. CONCLUSIONS The application of cyclic strain on seeded tendon constructs that were treated with the bioreactor helped achieve a UTS and an EM comparable with those of fresh tendons. Bioreactor pretreatment and alternative cell lines, such as ASCs and Fs, might therefore contribute to the in vitro production of strong tendon material.

Flexor Tendon Tissue Engineering: Temporal Distribution of Donor Tenocytes versus Recipient Cells

Background: Tissue-engineered tendon material may address tendon shortages in mutilating hand injuries. Tenocytes from rabbit flexor tendon can be successfully seeded onto acellularized tendons that are used as tendon constructs. These constructs in vivo exhibit a population of tenocyte-like cells; however, it is not known to what extent these cells are of donor or recipient origin. Furthermore, the temporal distribution is also not known. Methods: Tenocytes from New Zealand male rabbits were cultured and seeded onto acellularized rabbit forepaw flexor tendons (n = 48). These tendon constructs were transplanted into female recipients. Tendons were examined after 3, 6, 12, and 30 weeks using fluorescent in situ hybridization to detect the Y chromosome in the male donor cells. One unseeded, acellularized allograft in each animal was used as a control. Results: The donor male tenocytes populate the epitenon and endotenon of the grafts at greater numbers than the recipient female tenocytes at 3 and 6 weeks. The donor and recipient tenocytes are present jointly in the grafts until 12 weeks. At 30 weeks, nearly all cells are recipient tenocyte-like cells. Conclusions: Donor male cells survive in decreasing numbers over time until 30 weeks. The presence of cells in tissue-engineered tendon grafts has been shown in prior studies to add to the strength of the constructs in vitro. This study shows that recipient cells can migrate into and repopulate the tendon construct. Cell seeding onto tendon material may create stronger constructs that will allow the initiation of motion earlier.

Bioreactor optimization of tissue engineered rabbit flexor tendons in vivo

Tissue-engineered rabbit flexor tendons reseeded with cells are stronger in vitro after culture in a bioreactor. It is not known whether this effect persists in vivo. Tenocytes from New Zealand white rabbits were seeded onto rabbit rear paw flexor tendons that were deprived of cells and exposed to cyclic strain in a bioreactor. Reseeded constructs that were kept unloaded in a medium for 5 days were used as controls. The tendons were implanted to bridge a zone II defect in the rabbit. After explantation 4 weeks later, the ultimate tensile strength (UTS) and elastic modulus (EM) were determined. Tendon constructs that were exposed to cyclic strain had significantly improved UTS and EM. Histology showed that cellularity was increased in the bioreactor tendons.

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.

102A: BIOREACTOR OPTIMIZATION OF TISSUE ENGINEERED RABBIT FLEXOR TENDONS IN VIVO

Case: A 14 year-old male with Treacher-Collins syndrome presented with severe bi-orbitozygomatic hypoplasia confirmed by clinical examination and CT. Through a coronal incision, bilateral orbitozygomatic complexes were constructed using a combination of sculpted bone allograft, BMP-2, lipoasparite, and periosteal flaps. Follow-up clinical examination and CT scans were performed at regular intervals to evaluate bony competence. Pre-implant samples of ASCs and lipoaspirate underwent flow cytometric analysis.