Vaginal reconstruction using perioperative layered autologous tissue expansion, combined with self-assembly tissue engineering

Introduction: Tissue engineering has not been widely adopted in clinical settings for several reasons, including technical challenges, high costs, and regulatory complexities. The perioperative layered autologous tissue expansion (PLATE) technique is based on the principles of skin expansion used in dermatology and enables the reconstruction of hollow organs through tissue engineering in a single operation. It effectively addresses regulatory challenges associated with cell extraction. The PLATE technique is unique in that it can be performed in a standard operating room and is cost-effective.

Here we use the PLATE technique as an evaluation method for engineered tissue regeneration. Vaginal tissue was engineered with the self-assembly (SA) method, where fibroblasts produce a native like ECM that can be manipulated and seeded with epithelial cells to mature into a native-like tissue. SA-vaginal tissue was used in lieu of native biopsies for a comparison of the recolonization of the PLATE graft.

Materials and

Methods: SA-vaginal tissue was engineered by seeding primary vaginal fibroblasts at 1x10^6 cells/well in 6-well plates containing a Whatman paper ring and cultured with 50µM 2-phospho-L-ascorbic acid for 2 weeks. The wells were then re-seeded with the same density of vaginal fibroblasts and cultured 2 more weeks. 3 stromal sheets were stacked and fused for 24 hours in 60mm petri dishes. Primary vaginal epithelial cells were then seeded (1x10^6 cells/ tissue) and cultured for 1 week in submerged condition followed by 3 weeks at the air-liquid interface.

SA-vagina were then minced to 2x2 mm pieces and placed on a Vicryl™ mesh. Tissues were expanded 3X or 6X their original surface area. A collagen solution was poured over the Vicryl™ with minced tissue in a 2x6 cm mould, gelled for 10 minutes at 37°C, then plastically compressed under the weight of the mold to expel water from the gel and form a pliable construct containing entrapped tissue particles. The tissues were evaluated over 6 weeks of maturation in vitro, where some of the tissues were placed at the air-liquid interface after 3 weeks of culture. Fresh human vaginal mucosa tissue was used as a control in the PLATE method.

Histology was evaluated using Masson’s Trichrome staining and Periodic Acid-Schiff staining. The resulting epithelium was characterized by immunofluorescent labelling and immunohistochemistry.

Results, Conclusions, and Discussions: PLATE constructs using fresh vaginal biopsies formed a stratified squamous epithelium with a well-organized basal layer. The constructs appeared fully epithelialized after 3 weeks of culture. The epithelium thickened significantly after just 1 week of air-liquid culture. The stromal compartment showed significant reorganization over the 6 weeks of culture regardless of air-liquid condition. The collagen gel was colonized by fibroblasts and integrated with the native stroma.

When evaluating the SA-vaginal tissue, epithelial migration on the collagen gel was observed over the first 3 weeks of culture. In the following weeks, the tissue contracted significantly as the Vicryl™ mesh disintegrated, and the epithelium stopped proliferating, indicating a loss of potential. Interestingly, the SA-stroma did not integrate with the collagen gel.

Here, we see the therapeutic potential of the PLATE graft when used with native tissue biopsies. The epithelial cells are able to migrate to the gel surface and colonize the entire construct. The air-liquid condition is representative of in vivo conditions and indicates PLATE’s promising capacity to regenerate a native like histology.

These results indicate a lack of complexity of the SA-vagina to simulate wound healing in vitro. Proliferative capacity of the epithelial cells may be diminished following cell extraction and expansion before reconstruction.

Here, we have evaluated a more established tissue engineering method, SA, which has been in clinical trials for skin since 2014, compared to the novel PLATE technique which circumvents reglementary hurdles. These results underline the host’s role in integrating the lab grown tissues which lack in complexity, containing only 2 cell types. The PLATE’s natural complexity and untouched regenerative capacity may be at the origin of the impressive in vitro remodelling observed. Further evaluation is to be done to characterize in vivo integration of PLATE tissues. Potential drawbacks of the technique include a lack of native tissue for PLATE execution, or potential cyst formation during remodelling, as mucosal orientation is lost during the mincing step. Each model has its pros and cons and may be adapted to different situations.

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