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

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 fibrin-agarose 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 pulp-MSCs, 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 pre-differentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue. CONCLUSION: Our results suggest that the use of pre-differentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes.

Transversal Maxillary Distraction in Patients with Cleft Lip and Palate.

OBJECTIVE: The aim of this study is to describe the importance of osteodistraction with transpalatal distractors for treating transversal maxillary hypoplasia in patients with cleft and lip palate. METHODS: The participants were 17 patients (9 females and 8 males) with cleft lip and palate. Among these, 10 presented unilateral cleft lip and palate, 4 bilateral cleft lip and palate, and 3 cleft palate only. RESULTS: All patients experienced a satisfactory palatal expansion and crossbite correction. The mean lengthening was 12.7 mm. The average increase of intercanine distance, intermolar distance, maxillary transverse dimension (MTD), facial transverse dimension (FTD) was 12.16, 8.45, 1.77, and 1.67 mm, respectively. The clinical follow-up was 29.7 months (range: 6-61 months). CONCLUSION: Palatal distraction is a safe and successful alternative for treating maxillary transversal alterations in patients with cleft lip and palate. This technique facilitates the establishment of an adequate transverse dimension of maxillary, and consequently a proper dental occlusion.

Nasoalveolar Molding Therapy for the Treatment of Unilateral Cleft Lip and Palate Improves Nasal Symmetry and Maxillary Alveolar Dimensions.

OBJECTIVE: The aim of this study is to assess the esthetic and morphologic outcomes before surgery using nasoalveolar molding (NAM) therapy in children with unilateral cleft lip and palate. DESIGN: A prospective analysis was performed. SETTING: The study was carried out in the Congenital Malformations Craniofacial and Cleft Lip and Palate Unit, Hospital Virgen de las Nieves, Andalusian Health Service, Granada (Spain). PATIENTS: Twenty consecutively enrolled infants ranging in age from 7 to 30 days with nonsyndromic unilateral cleft lip and palate treated from 2008 to 2012. INTERVENTIONS: All patients were treated with NAM appliances to align the alveolar segments and reduce severity of the nasal deformity. MAIN OUTCOME MEASURE: The extraoral nasal measurements were performed on casts and nasal photographs. The measurements consisted of bialar width (BAW), columellar deviation (CD), cleft nostril height (CNH), cleft nostril width (CNW), non-CNH, non-CNW, and the deviation of the columella to the horizontal line represented by bilateral pupil line (BIA). The authors have made the measurements following Barilla method. Also 2 intraoral measurements were taken. RESULTS: Following NAM the extraoral records showed a statistically significant decrease in CD (P < 0.0001), CNW (P < 0.0001), and BAW (P < 0.001). Furthermore, statistically significant increases in CNH (P < 0.05) and BIA (P < 0.0001) were observed.Following Barilla measurements, the authors have found a high percentage of symmetry in all the nasal measurements after the NAM therapy.Intraoral results showed a statistically significant decrease in the gap between the greater and lesser alveolar segments and a statistically significant increase in maxillary arch width. CONCLUSIONS: Nasoalveolar molding improves nasal symmetry and achieves an improvement of all maxillary alveolar dimensions, increasing alveolar rim width, reducing the size of alveolar cleft gap, and improving shape of the maxillary dental arch. As a consequence of reducing the alveolar and nasal deformities before surgery, it is expected that the primary repair will be easier for the surgeon and more successful.

Histological characterization of the human masticatory oral mucosa. A histochemical and immunohistochemical study.

BACKGROUND: Histology of human oral mucosa is closely related with its function and anatomical location, and a proper characterization of the human masticatory oral mucosa could be very useful in periodontal pathology. OBJECTIVE: In the present work, we have carried out a comprehensive study in order to determine the main histological features of parakeratinized (POM) and orthokeratinized (OOM) masticatory human oral mucosa using light and electron microscopy. METHODS: To perform this, we have used several histological, histochemical and immunohistochemical methods to detect key markets at the epithelial, basement membrane and connective tissue levels. RESULTS: Our results demonstrated that POM and OOM share many histological similarities, as expected. However, important differences were observed at the epithelial layer of POM, that was significantly thicker than the epithelial layer found in OOM, especially due to a higher number of cells at the stratum spinosum. The expression pattern of CK10 and filaggrin revealed intense signal expression in OOM as compared to POM. Collagen and proteoglycans were more abundant in OOM stroma than in POM. No differences were found for blood vessels and basement membrane. CONCLUSION: These results may contribute to a better understanding of the pathological conditions affecting the human masticatory oral mucosa. In addition, these findings could be useful for the generation of different types of oral mucosa by tissue engineering techniques. RESEARCH HIGHLIGHTS: Microscopical features of parakeratinized and orthokeratinized masticatory human oral mucosa showed important differences at both, epithelial and stromal levels. Parakeratinized masticatory human oral mucosa exert thicker epithelial layer, especially, at the stratum spinosum in comparison to orthokeratinized human oral mucosa. Cytokeratin 10 and filaggrin human epithelial markers were intensively expressed in orthokeratinized masticatory human oral mucosa in comparison to parakeratinized masticatory human oral mucosa. At the stromal level, orthokeratinized masticatory human oral mucosa exhibit higher levels of collagen and proteoglycans than parakeratinized masticatory oral mucosa. The deep knowledge of histological features of masticatory oral mucosa could lead to a better understanding of oral mucosa pathology and advanced treatments.

Histological Profiling of the Human Umbilical Cord: A Potential Alternative Cell Source in Tissue Engineering.

The embryonic development of the human umbilical cord (hUC) is complex, and different regions can be identified in this structure. The aim of this work is to characterize the hUC at in situ and ex vivo levels to stablish their potential use in vascular regeneration. Human umbilical cords were obtained and histologically prepared for in the situ analysis of four hUC regions (intervascular-IV, perivascular-PV, subaminoblastic-SAM, and Wharton's jelly-WH), and primary cell cultures of mesenchymal stem cells (hUC-MSC) isolated from each region were obtained. The results confirmed the heterogeneity of the hUC, with the IV and PV zones tending to show the higher in situ expression of several components of the extracellular matrix (collagens, proteoglycans, and glycosaminoglycans), vimentin, and MSC markers (especially CD73), although isolation and ex vivo culture resulted in a homogeneous cell profile. Three vascular markers were positive in situ, especially vWF, followed by CD34 and CD31, and isolation and culture revealed that the region associated with the highest expression of vascular markers was IV, followed by PV. These results confirm the heterogeneity of the hUC and the need for selecting cells from specific regions of the hUC for particular applications in tissue engineering.

Usefulness of a Nanostructured Fibrin-Agarose Bone Substitute in a Model of Severely Critical Mandible Bone Defect.

Critical defects of the mandibular bone are very difficult to manage with currently available materials and technology. In the present work, we generated acellular and cellular substitutes for human bone by tissue engineering using nanostructured fibrin-agarose biomaterials, with and without adipose-tissue-derived mesenchymal stem cells differentiated to the osteogenic lineage using inductive media. Then, these substitutes were evaluated in an immunodeficient animal model of severely critical mandibular bone damage in order to assess the potential of the bioartificial tissues to enable bone regeneration. The results showed that the use of a cellular bone substitute was associated with a morpho-functional improvement of maxillofacial structures as compared to negative controls. Analysis of the defect site showed that none of the study groups fully succeeded in generating dense bone tissue at the regeneration area. However, the use of a cellular substitute was able to improve the density of the regenerated tissue (as determined via CT radiodensity) and form isolated islands of bone and cartilage. Histologically, the regenerated bone islands were comparable to control bone for alizarin red and versican staining, and superior to control bone for toluidine blue and osteocalcin in animals grafted with the cellular substitute. Although these results are preliminary, cellular fibrin-agarose bone substitutes show preliminary signs of usefulness in this animal model of severely critical mandibular bone defect.

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells.

Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin-agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results showed that the biofabrication methods used here succeeded in generating a tubular structure consisting of a stromal substitute with a stratified epithelial-like layer on top, especially using a medium containing epithelial growth and differentiation factors (EM), although differentiation was not complete. At the functional level, UM substitutes were able to synthesize collagen fibers, proteoglycans and glycosaminoglycans, although the levels of control UM were not reached ex vivo. Epithelial differentiation was partially achieved, especially with EM after 14 days of development, with expression of keratins 7, 8, and 13 and pancytokeratin, desmoplakin, tight-junction protein-1, and uroplakin 2, although at lower levels than controls. These results confirm the partial urothelial differentiative potential of HWJSC and suggest that the biofabrication methods explored here were able to generate a potential substitute of the human UM for future clinical use.

In Vitro Generation of Novel Functionalized Biomaterials for Use in Oral and Dental Regenerative Medicine Applications. Running Title: Fibrin-Agarose Functionalized Scaffolds.

Recent advances in tissue engineering offer innovative clinical alternatives in dentistry and regenerative medicine. Tissue engineering combines human cells with compatible biomaterials to induce tissue regeneration. Shortening the fabrication time of biomaterials used in tissue engineering will contribute to treatment improvement, and biomaterial functionalization can be exploited to enhance scaffold properties. In this work, we have tested an alternative biofabrication method by directly including human oral mucosa tissue explants within the biomaterial for the generation of human bioengineered mouth and dental tissues for use in tissue engineering. To achieve this, acellular fibrin-agarose scaffolds (AFAS), non-functionalized fibrin-agarose oral mucosa stroma substitutes (n-FAOM), and novel functionalized fibrin-agarose oral mucosa stroma substitutes (F-FAOM) were developed and analyzed after 1, 2, and 3 weeks of in vitro development to determine extracellular matrix components as compared to native oral mucosa controls by using histochemistry and immunohistochemistry. Results demonstrate that functionalization speeds up the biofabrication method and contributes to improve the biomimetic characteristics of the scaffold in terms of extracellular matrix components and reduce the time required for in vitro tissue development.

Expanded Differentiation Capability of Human Wharton's Jelly Stem Cells Toward Pluripotency: A Systematic Review.

Human Wharton's jelly stem cells (HWJSC) can be efficiently isolated from the umbilical cord, and numerous reports have demonstrated that these cells can differentiate into several cell lineages. This fact, coupled with the high proliferation potential of HWJSC, makes them a promising source of stem cells for use in tissue engineering and regenerative medicine. However, their real potentiality has not been established to date. In the present study, we carried out a systematic review to determine the multilineage differentiation potential of HWJSC. After a systematic literature search, we selected 32 publications focused on the differentiation potential of these cells. Analysis of these studies showed that HWJSC display expanded differentiation potential toward some cell types corresponding to all three embryonic cell layers (ectodermal, mesodermal, and endodermal), which is consistent with their constitutive expression of key pluripotency markers such as OCT4, SOX2, and NANOG, and the embryonic marker SSEA4. We conclude that HWJSC can be considered cells in an intermediate state between multipotentiality and pluripotentiality, since their proliferation capability is not unlimited and differentiation to all cell types has not been demonstrated thus far. These findings support the clinical use of HWJSC for the treatment of diseases affecting not only mesoderm-type tissues but also other cell lineages. Impact statement Human Wharton's jelly stem cells (HWJSC) are mesenchymal stem cells that are easy to isolate and handle, and that readily proliferate. Their wide range of differentiation capabilities supports the view that these cells can be considered pluripotent. Accordingly, HWJSC are one of the most promising cell sources for clinical applications in advanced therapies.

Erratum: Blanco-Elices, C. et al. In Vitro Generation of Novel Functionalized Biomaterials for Use in Oral and Dental Regenerative Medicine Applications. Running Title: Fibrin-Agarose Functionalized Scaffolds. Materials 2020, 13, 1692.

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[Alveoloplasty and the use of osteosynthesis material in the cleft lip palate]. / Alveoloplastias y utilización de material de osteosíntesis en la fisura labiopalatina.

OBJECTIVES: To compare the results of secondary alveoloplasty performed in one Hospital when osteosynthesis material was used and when the bone graft does not require this material, and relating them to factors such as gender and age. MATERIAL AND METHODS: A retrospective study was conducted from the years 2014 to 2019 in this Hospital on the selected patients who met the inclusion criteria. Two periods of ages, period A: ages between 5-12 years (mixed secondary alveoloplasty) and period B: greater than 12 years (late secondary alveoloplasty). Autologous bone from the iliac crest or parietal calotte was used for the bone graft. The patients were divided into 2 groups: group I: patients with alveoloplasties that required osteosynthesis material. Group II: patients who did not require osteosynthesis material. Parameters evaluated: the success criteria for alveoloplasty were assessed according to the clinical parameters described by Precious. Alveoloplasty was successful if they met all the criteria of Precious in the year of intervention. Postoperative complications in both groups were evaluated. The statistical analysis was performed using the exact Fisher test for qualitative variables. RESULTS: Alveoloplasty was successful in 89.4% of patients in group I, while it was 90.3% in group II. Alveoloplasty was successful in 87.5% of females compared to 91.17% of males. The intervention was a success in 91.48% of patients in group A, compared to 66.6% in group B. The osteosynthesis material in two patients of group I was not degraded in the annual assessment. There were no significant differences in any of the comparisons. CONCLUSIONS: The use of osteosynthesis material does not alter the integration of the bone graft in patients that undergo alveoloplasty. Factors such as gender or age do not influence the results of the interventions.

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

We evaluated the efficiency of several protocols to preserve the main components of decellularized tissue scaffolds for delayed use. Decellularized rat intestine scaffolds were generated by using SDS and triton X-100 and preserved for 3 months subjected to eight freeze-drying (F1 to F8) and 14 cryopreservation protocols (C1 to C14). Morphological analysis showed that cryopreservation tended to preserve the tissue morphostructure more efficiently than freeze-drying. Histological analysis showed that the content of proteoglycans and glycoproteins was efficiently preserved by most methods. The protocols that most efficiently preserved collagen fibers were those using trehalose and saccharose for freeze-drying (F2, F3, and F7 protocols) and DMSO, albumin, and saccharose (C3, C5, C6, C12) for cryopreservation. Most freeze-drying protocols and cryopreservation protocols with DMSO, albumin, and maltose (C6, C7, C13, and C14) efficiently preserved reticular fibers. For the elastic fibers, freeze-drying methods with trehalose and maltose (F2, F4, F6, and F8) properly preserved these fibers, with the results of most cryopreservation methods comparable to controls. These results suggest that freeze-drying using 0.1M trehalose and cryopreservation in the presence of 8% DMSO and 4.6% albumin are more efficient than other protocols in preserving the scaffold morphostructure and histological composition. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 488-500, 2018.

The IS2 Element Improves Transcription Efficiency of Integration-Deficient Lentiviral Vector Episomes.

Integration-defective lentiviral vectors (IDLVs) have become an important alternative tool for gene therapy applications and basic research. Unfortunately, IDLVs show lower transgene expression as compared to their integrating counterparts. In this study, we aimed to improve the expression levels of IDLVs by inserting the IS2 element, which harbors SARs and HS4 sequences, into their LTRs (SE-IS2-IDLVs). Contrary to our expectations, the presence of the IS2 element did not abrogate epigenetic silencing by histone deacetylases. In addition, the IS2 element reduced episome levels in IDLV-transduced cells. Interestingly, despite these negative effects, SE-IS2-IDLVs outperformed SE-IDLVs in terms of percentage and expression levels of the transgene in several cell lines, including neurons, neuronal progenitor cells, and induced pluripotent stem cells. We estimated that the IS2 element enhances the transcriptional activity of IDLV LTR circles 6- to 7-fold. The final effect the IS2 element in IDLVs will greatly depend on the target cell and the balance between the negative versus the positive effects of the IS2 element in each cell type. The better performance of SE-IS2-IDLVs was not due to improved stability or differences in the proportions of 1-LTR versus 2-LTR circles but probably to a re-positioning of IS2-episomes into transcriptionally active regions.

Generation of a bioengineered autologous bone substitute for palate repair: an in vivo study in laboratory animals.

We carried out an in vivo study to evaluate the potential usefulness of a novel bioengineered bone substitute for the repair of palate defects in laboratory rabbits, using tissue-engineering methods. Our results showed that the use of a bioengineered bone substitute was associated with more symmetrical palate growth as compared to the controls, and the length and height of the palate were very similar on both sides of the palate, with differences from negative controls 4 months after artificial bone grafting for bone length. The histological analysis revealed that the regenerated bone was well organized and expressed osteocalcin. In contrast, bone corresponding to control animals without tissue grafting was immature, with areas of osteoid tissue and remodelling, as determined by MMP-14 expression. These results suggest that bone substitutes may be a useful strategy to induce the formation of a well-structured palate bone, which could prevent the growth alterations found in cleft palate patients. This opens a door to a future clinical application of these bone substitutes. Copyright © 2015 John Wiley & Sons, Ltd.

Usefulness of a bioengineered oral mucosa model for preventing palate bone alterations in rabbits with a mucoperiostial defect.

The use of mucoperiostial flaps during cleft palate surgery is associated with altered palatal bone growth and development. We analyzed the potential usefulness of a bioengineered oral mucosa in an in vivo model of cleft palate. First, a 4 mm palate defect was created in one side of the palate oral mucosa of 3 week-old New Zealand rabbits, and a complete autologous bioengineered oral mucosa (BOM) or acellular fibrin-agarose scaffold (AS) was implanted. No material was implanted in the negative controls (NC), and positive controls were not subjected to palatal defect (PC). Animals were allowed to grow for 6 months and the results were analyzed morphologically (palate mucosa and bone size) and histologically. Results show that palatal mucosa and bone growth and development were significantly altered in NC and AS animals, whereas BOM animals had similar results to PC and the bioengineered oral mucosa was properly integrated in the host palate. The amount and compaction of collagen fibers was similar between BOM and PC, and both groups of animals had comparable contents of proteoglycans and glycoproteins at the palate bone. No differences were found for decorin, osteocalcin and BMP2. The use of bioengineered oral mucosa substitutes is able to improve palate growth and maturation by preventing the alterations found in animals with denuded palate bone. These results support the potential clinical usefulness of BOM substitutes for the treatment of patients with cleft palate and other conditions in which palate mucosa grafts are necessary with consequent bone denudation.

Histological and immunohistochemical changes in the rat oral mucosa used as an autologous urethral graft.

PURPOSE: The purpose of this study was to determine the histological and functional (immunohistochemical) changes that take place in oral mucosa grafts implanted in the rat urethra. METHODS: Urethroplasty was performed in 26 male Wistar rats weighing 250 g. All animals received autologous oral mucosa urethra grafting under general anesthesia. Samples were analyzed 10, 20, 30, 40, 50, 60, 90, and 120 days after surgery using light and scanning electron microscopy and immunofluorescence for the determination of the expression of epithelial markers (pancytokeratin, cytokeratin 1, 4, 13, and filaggrin). RESULTS: Grafted oral mucosa tissues were subjected to significant histological changes from the beginning with the formation of a well-developed epithelium whose structure was comparable to the native urethra from day 60 of the surgical implant. The immunofluorescence analysis demonstrated that the cytokeratin expression profile tended to mimic the pattern of the native urethra. These data suggest that the oral mucosa is able to efficiently transdifferentiate to the urethral environment. CONCLUSIONS: The efficient transdifferentiation process of the grafted oral mucosa at both the histological and immunofluorescence levels, and the absence of local complications confirm the clinical usefulness of this type of tissues for the repair of the urethra.