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. 2013 Oct;19(19-20):2242-52.
doi: 10.1089/ten.TEA.2012.0565. Epub 2013 Jun 25.

In vitro development and characterization of a tissue-engineered conduit resembling esophageal wall using human and pig skeletal myoblast, oral epithelial cells, and biologic scaffolds

Tigran Poghosyan  1 , Sebastien Gaujoux, Valerie Vanneaux, Patrick Bruneval, Thomas Domet, Severine Lecourt, Mohamed Jarraya, Rony Sfeir, Jerome Larghero, Pierre Cattan
Affiliations

In vitro development and characterization of a tissue-engineered conduit resembling esophageal wall using human and pig skeletal myoblast, oral epithelial cells, and biologic scaffolds

Tigran Poghosyan et al. Tissue Eng Part A. 2013 Oct.

Abstract

Introduction: Tissue engineering represents a promising approach for esophageal replacement, considering the complexity and drawbacks of conventional techniques.

Aim: To create the components necessary to reconstruct in vitro or in vivo an esophageal wall, we analyzed the feasibility and the optimal conditions of human and pig skeletal myoblast (HSM and PSM) and porcine oral epithelial cell (OEC) culture on biologic scaffolds.

Materials and methods: PSM and HSM were isolated from striated muscle and porcine OECs were extracted from oral mucosa biopsies. Myoblasts were seeded on an acellular scaffold issue from porcine small intestinal submucosa (SIS) and OEC on decellularized human amniotic membrane (HAM). Seeding conditions (cell concentrations [0.5×10(6) versus 10(6) cells/cm(2)] and culture periods [7, 14 and 21 days]), were analyzed using the methyl thiazoltetrazolium assay, quantitative PCR, flow cytometry, and immunohistochemistry.

Results: Phenotypic stability was observed after cellular expansion for PSM and HSM (85% and 97% CD56-positive cells, respectively), and OECs (90% AE1/AE3- positive cells). After PSM and HSM seeding, quantities of viable cells were similar whatever the initial cell concentration used and remained stable at all time points. During cell culture on SIS, a decrease of CD56-positive cells was observed (76% and 76% by D7, 56% and 70% by D14, 28% and 60% by D21, for PSM and HSM, respectively). Multilayered surface of α-actin smooth muscle and Desmine-positive cells organized in bundles was seen as soon as D7, with no evidence of cell within the SIS. Myoblasts fusion was observed at D21. Pax3 and Pax7 expression was downregulated and MyoD expression upregulated, at D14.OEC proliferation was observed on HAM with both cell concentrations from D7 to D21. The cell metabolism activity was more important on matrix seeded by 10(6) cells/cm(2). With 0.5×10(6) OEC/cm(2), a single layer of pancytokeratin-positive cells was seen at D7, which became pluristratified by D14, while when 10(6) OEC/cm(2) were used, a pluristratified epithelial structure was seen as soon as D7. Proliferative cells (Proliferating Cell Nuclear Antigen staining) were mainly located at the basal layer.

Conclusion: In this model, the optimal conditions of cell seeding in terms of cell concentration and culture duration were 0.5×10(6) myoblasts/cm(2) and 10(6) OEC/cm(2), and 7 days.

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Figures

FIG. 1.
FIG. 1.
(A) Small intestinal submucosa (SIS) (Hematoxylin Erythrosine Saffron [HES]×20); (B) Human amniotic membrane (HAM) with native epithelium (HES×20); (C) Decellularized HAM (HES×20). Color images available online at www.liebertpub.com/tea
FIG. 2.
FIG. 2.
Flow cytometry analysis of CD56, Desmine, and CD15 expression on human and porcine skeletal myoblasts (HSM and PSM) after cell expansion. Immunohistochemical staining (AE1/AE3) of porcine oral epithelial cell (OEC) after cell expansion. Color images available online at www.liebertpub.com/tea
FIG. 3.
FIG. 3.
Methyl thiazoltetrazolium (MTT) assay. Cell viability and proliferation analysis after seeding of two cell concentrations (0.5×106 cells/cm2 and 106 cells/cm2) and performed at three time points (D7, D14, and D21) (A) PSM: §, D0 versus D7 for both concentrations (p<0.001); &, D7 versus D21 (p=0.01 for 0.5×106cells/cm2 and p=0.04 for 106cells/cm2). (B) HSM: §, D0 versus D7 (p=0.028 for 0.5×106cells/cm2 and p<0.001 for 106cells/cm2); &, D7 versus D21 for 106 cells/cm2 (p=0.04). *, between the two cell concentrations at D7 (p=0.04).
FIG. 4.
FIG. 4.
Flow cytometry analysis of PSM phenotype: §, D0 versus D21 for CD56 (p<0.001) and Desmine (p=0.002) rates.
FIG. 5.
FIG. 5.
Flow cytometry analysis of HSM phenotype. (A) Mean rate of CD56-positive cells according to different culture conditions: §, D0 versus D21 for all conditions (p<0.001); & D21 (cells cultured on SIS) versus Day 28 (cells cultured at 60%–80% of confluence [p=0.025] and cells cultured in Petri dishes for 14 days at 60%–80% of confluence after extraction from SIS at D14 [p=0.03]). (B) Mean rate of Desmine-positive cells according different culture conditions: §, D0 versus D21 for all conditions (p<0.01); &, D21 (cells seeded on SIS) versus Day 28 (cells extracted from SIS by D14 and secondarily cultured in Petri dishes during 14 days at 60%–80% of confluence (p=0.034).
FIG. 6.
FIG. 6.
Expression of Pax3, Pax7, MyoG, and MyoD at D7 and D14. The 2−ΔΔCtvalues of Pax3, Pax7, MyoG, and MyoD at D0 are considerate as 1.
FIG. 7.
FIG. 7.
Pathology analysis. (A, B) HSMs on SIS by D7 (HES×20 and×40); (C, D) HSMs on SIS by D21 (HES×20 and×40); (E, F) HSMs on SIS by D7 (Desmine staining×20 and×40); (G, H) HSMs on SIS by D21. Multinucleate myotube. (Desmine staining×20 and×40). (I, J) PSMs on SIS by D7 (HES×10 and×40); (K, L) PSMs on SIS by D21 (HES×10 and×40); (M, N) PSMs on SIS by D7 (α-smooth muscle actin[SMA] staining×20); (O, P) PSMs on SIS by D21 (α-SMA staining×20). Due to the absence of difference between two concentrations, only scaffolds seeded by 0.5×106 cells/cm2 are shown. The arrows in (F) indicate desmin positive cells, the arrows in (H) indicate desmin positive cells and multinucleate myotube, and the arrows in (M–P) indicate SMA positive cells. Color images available online at www.liebertpub.com/tea
FIG. 8.
FIG. 8.
MTT assay. OEC viability and proliferation analysis after seeding of two cell concentrations (0.5×106 cells/cm2 and 106 cells/cm2) and performed at three time points (D7, D14, and D21): §, D0 versus D7 for both cell concentrations (p<0.001); &, D7 versus D21 for both cell concentrations (p=0.002 for 0.5×106 cells/cm2 and p<0.001 for 106 cells/cm2). *, between two cell concentrations within same time points (p<0.001).
FIG. 9.
FIG. 9.
Pathology analysis of porcine OEC on HAM (A) 0.5×106 cells/cm2, D7 (HES×20); (B) 0.5×106 cells/cm2, D14 (HES×10); (C) 106 cells/cm2, D7 (HES×10); (D) 106 cells/cm2, D14 (HES×10); (E) 0.5×106 cells/cm2, D14 (Proliferating Cell Nuclear Antigen [PCNA]×20); (F) 0.5×106 cells/cm2, D14 (pancytokeratin AE1/AE3×40); (G) 106 cells/cm2, D14 (PCNA×20); (H) 106 cells/cm2, D14 (pancytokeratin AE1/AE3×5). The arrows in (E,G) indicate PCNA positive cells. Color images available online at www.liebertpub.com/tea
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