Generation of a novel model of bioengineered human oral mucosa with increased vascularization potential

J Periodont Res. 2021;00:1–16. | 1wileyonlinelibrary.com/journal/jre Received: 7 July 2021 | Revised: 29 July 2021 | Accepted: 25 August 2021 DOI: 10.1111/jre.12927 ORIGINAL ARTICLE Generation of a novel model of bioengineered human oral mucosa with increased vascularization potential Cristina BlancoElices1,2,3 | Jesús ChatoAstrain1,2 | Salvador Oyonarte1,2,4 | Fabiola BermejoCasares1 | Antonio EspañaLópez5 | Ricardo FernándezValadés2,6 | Maria del Carmen SánchezQuevedo1,2 | Miguel Alaminos1,2 | Miguel Angel MartínPiedra1,2 | Ingrid Garzón1,2 This is an open access article under the terms of the Creat ive Commo ns Attri butio nNonCo mmerc ialNoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made. © 2021 The Authors. Journal of Periodontal Research published by John Wiley & Sons Ltd. 1Department of Histology (Tissue Engineering Group), University of Granada, Granada, Spain 2Instituto de Investigación Biosanitaria ibs. GRANADA, Granada, Spain 3Doctoral Programme in Biomedicine, University of Granada, Granada, Spain 4Andalusian Network for Transfusional Medicine, Cells and Tissues and Blood and Tissue Bank of Granada, Granada, Spain 5Craniofacial Malformations and Cleft Lip and Palate Management Unit, University Hospital Virgen de las Nieves, Granada, Spain 6Division of Pediatric Surgery, University Hospital Virgen de las Nieves, Granada, Spain Correspondence Miguel Angel MartínPiedra and Miguel Alaminos, Department of Histology, Faculty of Medicine, University of Granada. Avenida de la Investigación 11, E18016, Granada, Spain. Emails: [email protected] (MAMP) ; [email protected] (MA) Funding information Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I) of the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III), Grant/Award Number: FIS PI18/331, FIS PI21/00980, FIS PI18/332 and ICI19/00024; Consejería de Salud y Familias, Junta de Andalucía, Spain, Grant/Award Number: PI04422019; FEDER funds, European Union Abstract Objective: The aim of this study was to generate novel models of bioartificial human oral mucosa with increased vascularization potential for future use as an advanced therapies medicinal product, by using different vascular and mesenchymal stem cell sources. Background: Oral mucosa substitutes could contribute to the clinical treatment of complex diseases affecting the oral cavity. Although several models of artificial oral mucosa have been described, biointegration is a major issue that could be favored by the generation of novel substitutes with increased vascularization potential once grafted in vivo. Methods: Three types of mesenchymal stem cells (MSCs) were obtained from adipose tissue, bone marrow, and dental pulp, and their in vitro potential was evaluated by inducing differentiation to the endothelial lineage using conditioning media. Then, 3D models of human artificial oral mucosa were generated using biocompatible fibrinagarose biomaterials combined with human oral mucosa fibroblasts and each type of MSC before and after induction to the endothelial lineage, using human umbilical vein endothelial cells (HUVEC) as controls. The vascularization potential of each oral mucosa substitute was assessed in vitro and in vivo in nude mice. Results: In vitro induction of MSCs kept in culture was able to increase the expression of VEGF, CD31, and vWF endothelial markers, especially in bone marrow and dental pulpMSCs, and numerous proteins with a role in vasculogenesis become overexpressed. Then, in vivo grafting resulted in a significant increase in blood vessels formation at the interface area between the graft and the host tissues, with significantly positive expression of VEGF, CD31, vWF, and CD34 as compared to negative controls, especially when predifferentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed 2 | BLANCOELICES Et AL. 1 | INTRODUCTION Oral disorders are very prevalent conditions affecting more than 3 billion people worldwide, with oral cancer, trauma, and severe periodontal disease representing the most prevalent and severe cases.1 In most of these cases, treatment is highly dependent on the availability of healthy human oral mucosa for replacement therapy. In this regard, several models of threedimensional tissueengineered human oral mucosa have been developed, optimized, and characterized using different cells, biomaterials, and signaling molecules.2,3 Major applications of bioartificial oral mucosa include clinical transplantation in maxillofacial surgery, periodontics, and other related therapies.4 Although previously described models showed potential usefulness, one of the main clinical limitations of bioartificial tissues is the lack of vascularization, which could lead to tissue hypoxia once grafted in vivo, which could hinder biointegration.5 In this context, recent studies demonstrated the importance of generating bioartificial tissues containing a prevascular network capable of promoting an efficient supply of nutrients and oxygen after implantation.6 Different strategies have been developed to promote blood vessels formation in bioengineered tissues, including the incorporation of endothelial cells7, enriching biomaterials with endothelial growth factors8 or the direct injection of endothelial cells at the implantation site7, although none of these strategies demonstrated to be fully effective. Due to their demonstrated provasculogenic properties5, mesenchymal stem cells (MSCs) have been proposed as a promising strategy for vascularization of artificial tissues.9 Interestingly, MSCs have been previously used in tissue engineering due to their immunosuppressive and antiapoptotic properties able to favor regeneration of damaged tissues10 and their capability to act as potent regulators of the immune response.11 In addition, it has been shown that MSCs are able to express several angiogenic factors such as VEGF, which could facilitate a rapid vascularization of the tissue after implantation.12,13 Furthermore, it is well known that different types of MSCs of the human body are highly heterogeneous.14 For this reason, specific studies capable of determining the in vitro and in vivo vascularization potential of different MSCs sources used in bioengineered oral mucosa are in need. In addition, the provasculogenic role of human MSCs has not been previously demonstrated in maxillofacial and periodontal scenarios in which the use of MSCs for the generation of a human oral mucosa substitutes with improved vascularization potential could be an interesting approach. In this work, we have developed novel models of human artificial oral mucosa based on fibrinagarose biomaterials combined with MSCs able to differentiate to the endothelial cell lineage, in order to assess the possibility of inducing rapid vascularization and biointegration after in vivo implantation. 2 | MATERIALS AND METHODS 2.1 | Primary cell cultures 2.1.1 | Human oral mucosa fibroblasts (HFOM) To generate primary cell cultures of human oral mucosa fibroblasts (HFOM), small biopsies of human oral mucosa were obtained from healthy donors subjected to minor oral surgery with local anesthesia. In brief, human oral mucosa (HOM) fibroblasts were obtained by enzymatic digestion of the HOM stroma using 2 mg/ ml solution of Clostridium histolyticum type I collagenase (Gibco BRL) at 37ºC for 6 hours with agitation. Isolated HOM fibroblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (SigmaAldrich/Merck) supplemented with 10% fetal bovine serum (SigmaAldrich/Merck) and 1% antibiotics and antimycotics (100 U/ml penicillin G, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B) (SigmaAldrich/Merck) using 75 cm2 culture flasks with filter caps (Sarstedt, Nümbrecht, Germany), as previously described by the research group.15,16 2.1.2 | Mesenchymal stem cells (MSCs) To generate primary cultures of three types of human MSCs, we obtained small biopsies from adipose tissue, bone marrow, and dental pulp following previously described protocols.17,18,19,16 All tissue types were washed in phosphatebuffered saline (PBS) containing antibiotics and antimycotics and enzymatically digested for 6 h at 37ºC using a 2 mg/ml solution of Clostridium histolyticum type I collagenase (Gibco BRL). Isolated MSCs from adipose tissue (ADSC), bone marrow (BMSC), and dental pulp (DPSC) were cultured in 75 cm2 culture flasks with filter caps (Sarstedt) using DMEM supplemented with 10% fetal bovine serum and 1% antibiotics/antimycotics. In all cases, cells were cultured at 37ºC in a humidified incubator with 5% CO2 using standard cell culture to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue. Conclusion: Our results suggest that the use of predifferentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes. KEYWORDS mesenchymal stem cells, oral mucosa, tissue engineering, vascularization | 3 BLANCOELICES Et AL. conditions. The culture medium was changed every 2– 3 days. Once the cells reached 70% confluence, cells were dissociated with 0.25% trypsinethylenediaminetetraacetic acid (EDTA) (SigmaAldrich/Merck). 2.1.3 | Human umbilical vein endothelial cells (HUVEC) Human umbilical vein endothelial cells (HUVEC) were commercially obtained from the American Type Culture Collection (ATCC). HUVEC were cultured in Endothelial Growth Medium (SigmaAldrich/Merck) using 75 cm2 culture flasks with filter caps (Sarstedt). Cells were cultured at 37ºC in a humidified incubator with 5% CO2, and the culture medium was changed every 2– 3 days. 2.2 | MSCs characterization Multilineage differentiation potential of isolated MSCs was confirmed by subculturing MSCs on chamber slides and inducing differentiation to adipogenic, chondrogenic, and osteogenic cell lineages using specific conditioning media. Histochemical analysis was performed by oil red O, alizarin red S, and alcian blue as previously described.20 In addition, ADSC, BMSC, and DPSC were characterized to confirm their stemness profile before endothelial induction. For this, expression of typical MSCs markers was evaluated by flow cytometry using a Human MSC Analysis BD Stemflow™ kit (BD Biosciences). In brief, 5 × 105 cells corresponding to ADSC, BMSC, and DPSC were placed in flow cytometry tubes and washed with 2 ml of staining buffer (R&D Systems Inc). Then, Fc receptors were blocked by incubating the cells for 5 min with 2 ml of PBS containing 0.1% bovine serum albumin and 0.1% FBS. Next, cells were stained with a positive cocktail (FITC CD90, PerCPCy CD105, and APC CD73) and a negative MSC cocktail (PE CD45, PE CD34, PE CD11b, PE CD19, and PE HLADR) and incubated for 45 min at 4ºC in darkness. Once cells were labeled, cells were placed in a staining buffer and analyzed using a FACSCalibur flow cytometer (BD Biosciences). 2.3 | Endothelial differentiation of MSCs To induce the endothelial differentiation of nondifferentiated ADSC, BMSC, and DPSC (called ndADSC, ndBMSC, and ndDPSC, respectively), these cells were cultured for 21 days in an endothelial differentiation medium composed by 199 medium (SigmaAldrich/ Merck) supplemented with 20% FBS, 1.5% endothelial growth supplement from bovine neural tissue, and 1.5% antibiotics and antimycotics (100 U/ml penicillin G, 100 mg/ml streptomycin, and 0.25 mg/ ml amphotericin B) as previously reported.21 The culture medium was changed three times a week, and subconfluent cells were dissociated using 0.25% EDTA (all reagents, from SigmaAldrich/Merck). Cells induced with endothelial differentiation medium for 21 days were called differentiated cells (dADSC, dBMSC, and dDPSC, respectively). 2.4 | In vitro immunofluorescence analysis To identify the expression of relevant endothelial differentiation markers in ADSC, BMSC, and DPSC, we carried out immunofluorescence analyses using specific antibodies antivascular endothelial growth factor (VEGF), antiCD31 (PECAM1), antivon Willebrand Factor (vWF), and antiCD34. These analyses were carried out on native, nondifferentiated, and differentiated MSCs. HUVEC was used as a control group. First, 104 cells were cultured in chamber slides (NuncTM L a b - T e k TM Thermo Fisher Scientific), fixed with 70% ethanol, washed in PBS, and blocked for 30 min with normal horse serum and casein (Vector Laboratories). Then, samples were incubated for 1 h with primary antibodies with a dilution 1:100 for antiVEGF (Abcam), 1:500 for antiCD31 (PECAM1; Abcam), 1:100 for antivWF (Abcam), and 1:2500 for antiCD34 (Abcam), washed in PBS, and incubated with secondary antibodies for 1 h at room temperature using FITCconjugated antimouse (SigmaAldrich/Merck) or CY3conjugated antirabbit antibodies (SigmaAldrich/Merck). All study samples and controls were counterstained with DAPI (Vector Laboratories) and analyzed using an Eclipse 90i microscope (Nikon). Results were evaluated by three independent histologists using a semiquantitative method scoring the signal as strongly positive (+++), positive (++), mildly positive (+), slightly positive (+/−), or negative (−). 2.5 | Expression analysis of vascularizationrelated proteins Expression of relevant proteins playing a role in promoting vascularization was assessed in primary cell cultures of nondifferentiated and differentiated ADSC, BMSC, and DPSC using a human angiogenesis array kit (R&D Systems Inc.). First, 106 cells of each type were harvested and mixed with 1.0 ml of MCL1 mammalian cell lysis extraction buffer (SigmaAldrich/Merck) containing 20% of TrisEDTA, NaCl, SDS (lauryl sulfate), DOC (deoxycholic acid), Igepal CA, and 1% of protease inhibitor cocktail. Cell lysates were then centrifuged at 10,000 g for 10 min at 4ºC, and the supernatant was collected. Protein concentration was measured using a Pierce BCA200 Assay Kit (Thermo Fisher Scientific). Then, the protein array membranes were blocked with 2.0 ml of the buffer included in the kit, and 1.0 ml of protein extract corresponding to each type of sample was added to the membrane at final concentration of 300 µg/ml. After this, all samples were treated with 15 µl of reconstituted detection antibody cocktail, washed, and incubated with 2.0 ml of StreptavidinHRP. Finally, samples were washed and Chemi Reagent Mix was spread all over the membranes and incubated for one minute at room temperature prior to Xray film exposition for 10 min. Films were scanned, 4 | BLANCOELICES Et AL. and signal intensity was quantified at each spot using Image J software (Wayne Rasband, NIH). Analyses were carried out using triplicates (n = 3). 2.6 | Generation of novel models of human oral mucosa stroma with provascularization potential To determine the vascularization potential of each cell type in a threedimensional tissue substitute generated by tissue engineering, we generated novel models of human oral mucosa stroma (HOM) based on the previously described fibrinagarose biomaterial. In brief, hydrogels were developed with a mixture of human plasma combined with type VII agarose and cultured cells, as previously described.22,15 To prevent gel fibrinolysis, the mixture was supplemented with tranexamic acid Amchafibrin™ (Fides Ecopharma), and 1% calcium chloride was added at the final step to induce polymerization. This mixture was aliquoted on 6well plates with 24 mm Transwell Permeable Supports (Corning Life Sciences). Four different models were generated: 1) artificial oral mucosa stroma containing 5 × 105 oral mucosa fibroblasts (HOMHFOM). This is the previously developed bioartificial tissue and was used as negative control, 2) artificial oral mucosa stroma containing 2.5 × 105 oral mucosa fibroblasts and 2.5 × 105 HUVEC (HOMHUVEC). This tissue substitute was used as positive control; 3) artificial oral mucosa stroma containing 2.5 × 105 oral mucosa fibroblasts and 2.5 × 105 nondifferentiated MSCs (HOMndADSC, HOMndBMSC, or HOMndDPSC); and 4) artificial oral mucosa stroma containing 2.5 × 105 fibroblasts and 2.5 × 105 differentiated MSCs (HOMdADSC, HOMdBMSC, or HOMdDPSC) (Figure 1). 2.7 | In vivo evaluation of bioengineered human oral mucosa models To determine the in vivo vascularization potential of each model of bioengineered oral mucosa stroma, HOM models were grafted on immunedeficient Foxn1nu−/Foxn1nu− athymic mice. Shortly, mice were anesthetized using a mixture of 0.001 mg/g of body weight of acepromazine— CalmoNeosan™— (Boehringer Ingelheim,) and 0.15 mg/g of body weight of ketamine— Imalgene 1000— (Merial labs). Then, a skin area of 24 mm2 was surgically removed from the interscapular area of each animal and the bioengineered oral mucosa stroma models were trimmed to the same diameter. Samples were grafted at the injury site using absorbable suture material, and a plastic ring was implanted and sutured at the injury borders to prevent border contraction. All mice were euthanized one week after implantation of the bioengineered tissue for histological and immunohistochemical analyses. 2.8 | Histology and immunohistochemistry Tissues were fixed in 4% formaldehyde and embedded in paraffin in order to obtain 5 μmthick histological sections. Tissue sections were dewaxed, rehydrated, and stained with hematoxylin and eosin (HE) for histological analysis. First, the localization and distribution of the human cells grafted in the animals were determined by immunohistochemistry for the antihuman mitochondriaspecific antigen using antiMitochondria clone 113– 1 antibodies (SigmaAldrich/Merck), using a 1:80 dilution of the primary antibody. Then, to evaluate the in vivo vascularization potential of each HOM model, an immunohistochemical analysis was performed using the wellestablished markers of endothelial cells and blood vessels23,24 antiVEGF (Abcam) with 1:100 dilution, antiCD31 (PECAM1, Abcam) with 1:500 dilution, antivWF (Abcam) with 1:100 dilution, and antiCD34 with 1:2500 dilution as were previously used for in vitro analysis. In all cases, tissue sections were deparaffinized using xylene and rehydrated through a graded series of alcohols (100%, 96%, 70%, 50%, and water) before antigen retrieval with 0.01 M citrate buffer, pH 6.0, and 0.01 M EDTA buffer, pH 8.0, at 95ºC. Then, samples were prehybridized for 30 min with normal horse serum and casein (Vector Laboratories). Then, samples were incubated overnight with primary antibodies at 4ºC, washed with PBS, and incubated with secondary antibodies for 1 h at room temperature. The complex epitopeantibody was detected using 3,3diaminobenzidine - DAB- (Vector Laboratories), and tissue sections were counterstained for 20 s using Harry's hematoxylin. In all cases, images were obtained using a Pannoramic® DESK II DW scanner (3D Histotech). FIGURE 1 Schematic representation of the experimental groups used in the present work. (A) In vitro control group of human umbilical vein endothelial cells (HUVEC) kept in culture. (B) In vitro nondifferentiated MSCs (ndMSCs), including adipose stem cells (ndADSC), bone marrow stem cells (ndBMSC), and dental pulp stem cells (ndDPSC) kept in culture. (C) In vitro differentiated MSCs (dMSCs), including adipose stem cells (dADSC), bone marrow stem cells (dBMSC), and dental pulp stem cells (dDPSC) kept in culture. (D) Generation of negative control bioartificial human oral mucosa substitutes (HOMHFOM) containing oral mucosa fibroblasts (hOM Fibroblasts). (E) Generation of positive control bioartificial human oral mucosa substitutes (HOMHUVEC) containing human umbilical vein endothelial cells (HUVEC) and hOM Fibroblasts. (F) Generation of bioartificial human oral mucosa substitutes (HOMndMSCs) containing nondifferentiated MSCs and hOM Fibroblasts. (G) Generation of bioartificial human oral mucosa substitutes (HOMdMSCs) containing differentiated MSCs and hOM Fibroblasts. (H) Immunohistochemical analysis of antihuman Mitochondria in bioartificial human oral mucosa substitutes implanted on athymic mice. Peripheric areas of the injury site corresponding to mouse host tissue are squared in green. Human oral mucosa substitutes implanted on the athymic mice are squared in orange. Higher magnification images showing human origin cells (in brown) and host mouse cells (in blue), along with blood vessels showing positive signal (white arrows) and negative signal (black arrows) are shown in panels to the right. Scale bars: 100 µm | 5 BLANCOELICES Et AL. For each vascular marker, microvessel density (MVD) was measured by quantifying the number of blood vessels per mm2 of tissue surface at the vascular interphase area of each sample type. In addition, the number of cells showing positive signal was also quantified at the cellular area of the grafted tissues to determine the presence of cells committed to the endothelial lineage. 6 | BLANCOELICES Et AL. 2.9 | Statistical analysis To analyze the protein expression results obtained with the human angiogenesis array kit, we first determined the average expression found for each type of sample and calculated the foldchange value of differentiated cells vs. nondifferentiated cells of the same cell type (for instance, dADSC vs. ndADSC). Then, a rank test was applied to compare the results obtained for differentiated cells vs. nondifferentiated cells of the same cell type (for instance, dADSC vs. ndADSC). Proteins showing 1) an increase of at least 20% expression upon differentiation (foldchange >1.20) and 2) a p value below 0.05 for the rank test were selected as statistically significant. For the analysis of vascular markers, quantification results of blood vessels or cells showing positive signal for each marker were compared with negative control HOMHFOM and positive control HOMHUVEC using the Pairwise MannWhitney exact test, since samples did not fulfill normality criteria. p values below.05 were considered statistically significant, and tests were performed doubletailed. These comparisons were carried out using the Real Statistics Resource Pack software (Release 7.2) available at www.realstati stics.com. 2.10 | Research committee approval and ethics statement This study and the donation of the tissues used in this work (oral mucosa and tissues rich in MSC) were approved by the Research and Ethics Committee in Biomedical Research of Granada (Comité Coordinador de Ética de la Investigación Biomédica, ref. 0116N19, approval date 29/05/2019). All tissue donors provided informed consent. Animal experimentation was approved by the Animal Experimentation Ethics Committee of Granada (Comité de Ética y Experimentación Animal, CEEA) and Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible, Junta de Andalucía, Spain, protocol code 08/07/2019/123, date of approval January, 10/09/2019. 3 | RESULTS 3.1 | Phenotype characterization of human MSCs Histological evaluation of MSCs cultures (ADSC, BMSC, and DPSC) demonstrated that these cells were able to adhere to the culture flask surface, and the typical mesenchymal spindleshape, elongated morphology. Specific conditioning media revealed that MSCs used in this study were able to differentiate into adipogenic, chondrogenic, and osteogenic cell lineages, as determined by alizarin red S, alcian blue, and oil red O staining (Figure 2). Flow cytometry characterization of native MSCs cultures demonstrated that ndADSC, ndBMSC, and ndDPSC were positive for CD73, CD90, and CD105 markers. The analysis of CD73 marker showed 99.7% positive cells in ADSC, 95.4% in BMSC, and 99.3% in DPSC, whereas CD90 was positive in 85.2% of ndADSC, 87.0% of ndBMSC, and 97.8% of ndDPSCS, and CD105 was positive in 84.8% ndADSC, 89.7% ndBMSC, and 97.3% ndDPSC. Negative cocktail markers (CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLADR) were absent in all mesenchymal stem cell types (Figure 2). 3.2 | In vitro differentiation of human MSCs 3.2.1 | Immunofluorescence assay Semiquantitative analysis of several markers of endothelial differentiation revealed that HUVEC expressed VEGF, CD31, and vWF, but were negative for CD34. In the case of nondifferentiated MSCs, most markers were negative, except for ndBMSC, which had mildly positive expression of VEGF and vWF. Moreover, ndDPSC revealed slightly positive vWF expression. When MSCs were induced in vitro, we found an increase in the expression of VEGF, CD31, and vWF. For VEGF, we found a mildly positive signal in dADSC and a positive signal in dBMSC and dDPSC, whereas CD31 was slightly positive in dBMSC, and dDPSC and vWF showed strongly positive signal in dADSC and positive signal in dBMSC and dDPSC, and CD34 was negative in all cell types (Figure 3; Table 1). 3.2.2 | Protein expression analysis Several proteins became upregulated in cells subjected to in vitro induction. As shown in Figure 4, 11 out of the 55 proteins analyzed (20%) were upregulated in dDPSC, including Amphiregulin, Artemin, EGVEGF, Endoglin, FGF basic, HGF, IGFBP2, IGFBP3, LAP (TGFβ1), Platelet Factor 4 (PF4), and VEGF, with three of these proteins (Amphiregulin, PF4, and VEGF) showing more than 100fold expression upon differentiation. Eight proteins (14.5%) were significantly upregulated in dBMSC, including CXCL16, Coagulation Factor III, EGF, FGF4, LAP (TGFβ1), Serpin F1, TIMP1, and TIMP4. Interestingly, 6 of the 8 proteins upregulated in dBMSC were also found to be overexpressed in differentiated dADSC, which showed 10 proteins (18.2%) upregulated upon induction, including CXCL16, E G F , F G F - 4 , I L - 1 β, PF4, Serpin F1, Thrombospondin2, TIMP1, TIMP4, and Vasohibin. 3.3 | In vivo evaluation of the vascularization potential of the novel models of HOM 3.3.1 | Histological and immunohistochemical analysis All artificial HOM models grafted in vivo were properly biointegrated in the host animal, and we did not find any microscopical signs of hemorrhage, tumorigenesis, rejection, infection, or other side effects linked to the grafted tissues. | 7 BLANCOELICES Et AL. FIGURE 2 Phenotype characterization of human MSCs. (A) Multilineage differentiation capabilities of ADSC, BMSC, DPSC after adipogenic, chondrogenic, and osteogenic induction Scale bars: 100 µm. (B) Evaluation of the stemness profile of MSCs by the detection of CD73, CD90, and CD105 markers by flow cytometry 8 | BLANCOELICES Et AL. As shown in Figure 5, histological analysis of grafted HOM revealed the presence of three histological zones at the grafting site (zones A, B, and C). Zone A corresponded to the remaining artificial oral mucosa stroma substitute implanted in the animals. Zone B corresponded to the rest of the grafted HOM containing abundant cells of the bioartificial tissue. Finally, zone C represented the interface area between the native tissues belonging to the host animals and the grafted HOM, and contained numerous blood vessels. Interestingly, cells tended to concentrate at specific zones, especially at zone B, followed by zone C, while very few cells were found in zone A. In order to confirm the human origin of HFOM, HUVEC, and MSCs implanted in mice, samples were analyzed using antihuman mitochondria antigen antibodies. Results revealed a positive expression in the grafted tissue, whereas peripheral host tissues showed negative signal (Figure 1 and supplementary Figure S1). 3.3.2 | Analysis of vascular markers by Immunohistochemistry To determine the vascularization potential of the novel models of HOM, we analyzed the presence of structures showing positive signal for each specific marker in each tissue type grafted in vivo (Figures 610). In general, zone A was negative for the analyzed markers, whereas zone B contained numerous positive cells and zone C was rich in vessels showing positive immunohistochemical staining and was used to determine MVD. In addition, the use of FIGURE 3 In vitro characterization of human umbilical vein endothelial cells (HUVEC), nondifferentiated MSCs (ndMSCs) and differentiated MSCs (dMSCs) using CD34, VEGF, CD31 and vWF immunofluorescence. ndADSC: nondifferentiated adipose stem cells; ndBMSC: nondifferentiated bone marrow stem cells; ndDPSC: nondifferentiated dental pulp stem cells; dADSC: differentiated adipose stem cells; dBMSC: differentiated bone marrow stem cells; dDPSC differentiated dental pulp stem cells. Scale bars: 50 µm TABLE 1 Semiquantitative analysis of relevant vascular markers in human umbilical vein endothelial cells (HUVEC), nondifferentiated MSCs (ndMSCs), and MSCs differentiated in vitro (dMSCs). Expression was classified as strongly positive (+++), positive (++), mildly positive (+), slightly positive (+/−), or negative (−) HUVEC ndADSC ndBMSC ndDPSC dADSC dBMSC dDPSC VEGF +−+−+++ ++ CD31 +− − − − +/− +/− vWF +++ −++/− +++ ++ ++ CD34 − − − − − − − | 9 BLANCOELICES Et AL. antihuman mitochondria antigen antibodies allowed us to find that some of these blood vessels, approximately half of them, were of human origin, whereas other vessels consisted of host mouse cells (Figure 1). First, analysis of MVD in zone C using VEGF markers (Figures 6 and 10) showed that the number of vessels found in HOMHUVEC positive controls was significantly higher than HOMHFOM negative controls (p = 0.0188). For HOM generated with nondifferentiated MSCs, only HOMndBMSC showed lower MVD than positive controls (p = 0.0106), while HOMndDPSC was significantly higher than negative controls (p = 0.0244). HOMndADSC, HOMndDPSC, and all HOM tissues generated with differentiated cells were comparable to positive controls (p > 0.05 for all of them). Interestingly, MVD was significantly higher in HOMdBMSC as compared to HOMndBMSC (p = 0.0188) and in HOMdDPSC as compared to HOMndDPSC (p = 0.0244). When VEGFpositive cells were quantified in zone B, we found very few positive cells in HOMHFOM negative controls and in HOMndDPSC and very high number in HOMHUVEC positive controls, with differences being statistically significant (p < 0.0001 for HOMHFOM vs. HOMHUVEC and p = 0.0001 for HOMndDPSC vs. HOMHUVEC). HOM generated with nondifferentiated MSCs showed significant differences with positive controls (p = 0.0019 for HOMndADSC, p < 0.0001 for HOMndBMSC and p = 0.0001 for HOMndDPSC), and HOMndADSC (p = 0.0019) and HOMndBMSC (p = 0.0142) were also significantly higher than negative controls. Finally, HOM containing differentiated MSCs showed statistical differences with negative controls (p < 0.0001 for HOMdADSC and HOMdDPSC and p = 0.0019 for HOMdBMSC), but were comparable to positive controls (p > 0.05) and were significantly higher than HOM containing nondifferentiated MSCs (p = 0.0019 for HOMndADSC vs HOMdADSC and for HOMndBMSC vs HOMdBMSC, and p < 0.0001 for HOMndDPSC vs. HOMdDPSC). For CD31 (Figures 7 and 10), we found that the MVD in zone C was significantly lower in negative controls (p = 0.0002) and HOM generated with nondifferentiated MSCs (p < 0.0001 for HOMndADSC, p = 0.0008 for HOMndBMSC, and p = 0.0002 for HOMndDPSC) than in positive controls, with nonsignificant differences among these samples. For HOM containing differentiated MSCs, H O M - d A D S C ( p = 0.0028) and HOMdDPSC (p = 0.0106) contained significantly lower number of vessels than positive controls, although HOMdBMSC was similar to positive controls (p > 0.05). HOMdBMSC and HOMdDPSC were significantly higher than negative controls (p = 0.0106 and p = 0.0142, respectively). HOMdADSC and HOMdDPSC showed significantly higher MVD than HOMndADSC (p = 0.0003) and HOMndDPSC (p=0.0188). At zone B, the number of CD31positive cells was significantly higher in positive controls than in negative controls (p < 0.0001). HOM containing nondifferentiated MSCs had significantly fewer cells than positive controls (p < 0.0001 for all comparisons), although HOMndBMSC and HOMndDPSC were significantly higher than negative controls (p < 0.0001). For differentiated HOM, we found a significant increase in positive cells, and HOMdBMSC and HOMdDPSC became comparable to positive controls (p > 0.05). Analysis of MVD as determined by vWF immunostaining of vessels found in zone C (Figures 8 and 10) revealed again that the number of vWFpositive cells was higher in positive controls as compared to negative controls (p < 0.0001). 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Generation of a novel model of bioengineered human oral mucosa with increased vascularization potential. J Periodont Res. 2021;00:1– 16. https://doi.org/10.1111/jre.12927