Biochemical Indicators of Implantation Success of Tissue-Engineered Oral Mucosa

Abstract

Real-time (RT) determination of the health of in vitro tissue-engineered constructs prior to grafting is essential for prediction of success of the implanted tissue-engineered graft. In addition, the US Food and Drug Administration requires specific release criteria in RT prior to the release of tissue-engineered devices for human use. In principle, assessing the viability and functionality of the cellular component can be achieved by quantifying the secretion of growth factors and chemokines of tissue-engineered constructs. Ex vivo-produced oral mucosa equivalents (EVPOMEs) were fabricated under thermally stressed conditions at 43 °C for 24 h to create a functionally compromised EVPOME. We used microchannel enzyme-linked immunosorbent assay to evaluate the functionality of the cellular component, oral keratinocytes, of stressed and unstressed EVPOMEs by measuring the release of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), human β-defensin 1 (hBD-1), and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and -2) into the spent medium, which was collected on the same day prior to graft implantation into severe combined immunodeficiency mice. Implanted EVPOMEs' histology on the seventh postimplantation day was used to correlate outcomes of grafting to secreted amounts of IL-8, hBD-1, VEGF, TIMP-1, and TIMP-2 from corresponding EVPOMEs. Our findings showed that significantly higher levels of IL-8, hBD-1, and TIMP-2 were secreted from controls than from thermally stressed EVPOMEs. We also found a direct correlation between secreted VEGF and IL-8 and blood vessel counts of implanted EVPOMEs. We concluded that measuring the constitutive release of these factors can be used as noninvasive predictors of healthy tissue-engineered EVPOMEs in RT, prior to their implantation.

Living cell‐based regenerative medicine technologies for periodontal soft tissue augmentation

“…Khmaladze and coworkers recently proposed a non‐invasive method that allows real‐time monitoring of the thermal stress, and therefore the viability, of the EVPOME before implantation . The same group demonstrated that high levels of interleukin‐8 (IL‐8), human β‐defensin I (hBD‐I) and tissue inhibitor of metalloproteinase 2 (TIMP‐2) were predictors of healthy EVPOME . Nevertheless, further clinical studies are needed, as this method appears promising not only for distinguishing stress and non‐stressed EVPOME before implantation but also for evaluating post‐grafted outcomes …”

Comparison of two decellularized dermal equivalents

“…Allopatch scaffolds were rehydrated in DPBS for 1.5 hr in the initial protocol but were rehydrated in DPBS overnight for optimized procedures to manufacture EVPOMEs using Allopatch based on the results of the diffusion pattern analysis. The manufacturing of EVPOMEs was reported previously (Izumi et al, ; Izumi, Song, & Feinberg, ; Kuo et al , ). Briefly, 200,000 oral keratinocytes/cm 2 were seeded on 1 cm‐diameter acellular Allopatch and AlloDerm® scaffolds that were presoaked in 0.05 μg/μl human Type IV collagen (Sigma‐Aldrich, St. Louis, MO, USA).…”

“…The University of Michigan Institutional Review Board approved all procedures of harvesting human oral mucosal and skin tissues. The procedures for culturing primary human oral and skin keratinocytes were described previously (Bayar et al, ; Kuo et al, ). Briefly, primary human oral and skin keratinocytes were enzymatically dissociated using 0.04% and 0.125% trypsin (Sigma‐Aldrich, St. Louis, MO, USA), respectively, from the tissue samples, and cell cultures were established in serum free chemically defined culture medium (EpiLife and EDGS, Life Technologies, Grand Island, NY, USA) containing 0.06 mM calcium, 25 μg/ml gentamicin, and 0.375 μg/ml fungizone.…”

“…Mouse surgical procedures were described previously (Kuo et al , ; Izumie et al, 2003). Ten AlloDerm® and 10 Allopatch EVPOMEs were grafted into 7‐ to 8‐week‐old SCID mice (Charles River Laboratories, Wilmington, MA, USA).…”

“…In our laboratory, AlloDerm® has been used to manufacture ex vivo tissue‐engineered oral mucosa products (Khmaladze et al, ; Kuo et al, ), but Allopatch has not. In this report, we evaluated how cells cultured on AlloDerm® and Allopatch differ in terms of cellular growth and differentiation using ex vivo produced oral mucosa equivalents (EVPOMEs) as a study model (Izumi, Takacs, Terashi, & Feinberg, ; Kuo et al, ). To manufacture an EVPOME, oral keratinocytes are seeded at a high density so that the scaffold surface will be oversaturated with cells.…”