Development of a multilayered palate substitute in rabbits: a histochemical ex vivo and in vivo analysis

Abstract

Current tissue engineering technology focuses on developing simple tissues, whereas multilayered structures comprising several tissue types have rarely been described. We developed a highly biomimetic multilayered palate substitute with bone and oral mucosa tissues using rabbit cells and biomaterials subjected to nanotechnological techniques based on plastic compression. This novel palate substitute was autologously grafted in vivo, and histological and histochemical analyses were used to evaluate biointegration, cell function, and cell differentiation in the multilayered palate substitute. The three-dimensional structure of the multilayered palate substitute was histologically similar to control tissues, but the ex vivo level of cell and tissue differentiation were low as determined by the absence of epithelial differentiation although cytokeratins 4 and 13 were expressed. In vivo grafting was associated with greater cell differentiation, epithelial stratification, and maturation, but the expression of cytokeratins 4, 13, 5, and 19 at did not reach control tissue levels. Histochemical analysis of the oral mucosa stroma and bone detected weak signals for proteoglycans, elastic and collagen fibers, mineralization deposits and osteocalcin in the multilayered palate substitute cultured ex vivo. However, in vivo grafting was able to induce cell and tissue differentiation, although the expression levels of these components were always significantly lower than those found in controls, except for collagen in the bone layer. These results suggest that generation of a full-thickness multilayered palate substitute is achievable and that tissues become partially differentiated upon in vivo grafting.

Generation and Evaluation of Novel Stromal Cell‐Containing Tissue Engineered Artificial Stromas for the Surgical Repair of Abdominal Defects

“…Animals were deeply anesthetized and biopsies of the dorsal skin were obtained. Cell isolation and culture were performed as previously described . Briefly, tissues were trimmed and washed in phosphate‐buffered saline and digested in 2 mg mL −1 type I collagenase (Gibco, Karlsruhe, Germany) at 37° C to isolate the tissue SC.…”

In Vitro Generation of Novel Functionalized Biomaterials for Use in Oral and Dental Regenerative Medicine Applications

“…Previously reported human oral mucosa stroma substitutes based on fibrin-agarose biomaterials (n-FAOM) were generated following previously published methods [13,15]. Briefly, human oral mucosa biopsies were obtained from healthy donors subjected to minor oral surgery with local anesthesia at the School of Dental Sciences of the University of Granada, Spain.…”

“…In this milieu, one of the main limitations of tissue engineering is the need of obtaining adequate primary cell cultures generated from small tissue biopsies, which are later combined with biomaterials to generate a tissue substitute. In oral cavity and dental tissue engineering, biopsies are typically obtained from patient's oral mucosa, palate, periodontal tissue, or tooth, which are enzymatically digested to isolate an initial cell population that is subsequently expanded for further use in tissue engineering [13]. The fact that primary cell cultures show very low expansion and proliferation rate is one of the major factors associated to the long time required for the efficient generation of a human tissue substitute by tissue engineering [14] and supports the search for alternative strategies.…”

Evaluation of freeze‐drying and cryopreservation protocols for long‐term storage of biomaterials based on decellularized intestine

“…One of the possible limitations of the present study is the quantification method used to determine the amount of ECM constituents of the different samples. Although this is a simple straightforward method that has been extensively used by our research group, 4 , direct quantification of the stained areas could preferentially reflect the mean signal intensity of positively stained areas as suggested by Gonzalez and cols, thus failing to accurately report ECM components preservation after treatment in the whole tissue. Therefore, it could be used as an indicator of the retained portion of a specific ECM component after preservation.…”