Tissue-engineered Oral Mucosa Research Articles

Impact on postoperative oral morbidity and mid-term efficacy of anterior urethroplasty using an autologous tissue-engineered oral mucosa graft (MukoCell®) versus native oral mucosa graft

Impact on postoperative oral morbidity and mid-term efficacy of anterior urethroplasty using an autologous tissue-engineered oral mucosa graft (MukoCell®) versus native oral mucosa graft

In-process monitoring of a tissue-engineered oral mucosa fabricated on a micropatterned collagen scaffold: use of optical coherence tomography for quality control

BackgroundWe previously reported a novel technique for fabricating dermo-epidermal junction (DEJ)-like micropatterned collagen scaffolds to manufacture an ex vivo produced oral mucosa equivalent (EVPOME) for clinical translation; however, more biomimetic micropatterns are required to promote oral keratinocyte-based tissue engineering/regenerative medicine. In addition, in-process monitoring for quality control of tissue-engineered products is key to successful clinical outcomes. However, evaluating three-dimensional tissue-engineered constructs such as EVPOME is challenging. This study aimed to update our technique to fabricate a more biomimetic DEJ structure of oral mucosa and to investigate the efficacy of optical coherence tomography (OCT) in combination with deep learning for non-invasive EVPOME monitoring. MethodsA picosecond laser-textured microstructure mimicking DEJ on stainless steel was used as a negative mould to fabricate the micropatterned collagen scaffold. During EVPOME manufacturing, OCT was applied twice to monitor the EVPOME and evaluate its epithelial thickness. FindingsOur moulding system resulted in successful micropattern replication on the curved collagen scaffold. OCT imaging visualised the epithelial layer and the underlying micropatterned scaffold in EVPOME, enabling to non-invasively detect specific defects not found before the histological examination. Additionally, a gradual increase in epithelial thickness was observed over time. ConclusionThese findings demonstrate the feasibility of using a stainless-steel negative mould to create a more biomimetic micropattern on collagen scaffolds and the potential of OCT imaging for quality control in oral keratinocyte-based tissue engineering/regenerative medicine.

Fabrication of Micropatterned Fish Scale Collagen Scaffold Using Microelectromechanical Systems Technologies for Oral Mucosa Tissue Engineering

Fabrication of Micropatterned Fish Scale Collagen Scaffold Using Microelectromechanical Systems Technologies for Oral Mucosa Tissue Engineering

In-Process Monitoring of a Tissue-Engineered Oral Mucosa Fabricated on a Micropatterned Collagen Scaffold: Use of Optical Coherence Tomography for Quality Control

In-Process Monitoring of a Tissue-Engineered Oral Mucosa Fabricated on a Micropatterned Collagen Scaffold: Use of Optical Coherence Tomography for Quality Control

Cells/colony motion of oral keratinocytes determined by non-invasive and quantitative measurement using optical flow predicts epithelial regenerative capacity

Cells/colony motion determined by non-invasive, quantitative measurements using the optical flow (OF) algorithm can indicate the oral keratinocyte proliferative capacity in early-phase primary cultures. This study aimed to determine a threshold for the cells/colony motion index to detect substandard cell populations in a subsequent subculture before manufacturing a tissue-engineered oral mucosa graft and to investigate the correlation with the epithelial regenerative capacity. The distinctive proliferating pattern of first-passage [passage 1 (p1)] cells reveals the motion of p1 cells/colonies, which can be measured in a non-invasive, quantitative manner using OF with fewer full-screen imaging analyses and cell segmentations. Our results demonstrate that the motion index lower than 40 μm/h reflects cellular damages by experimental metabolic challenges although this value shall only apply in case of our culture system. Nonetheless, the motion index can be used as the threshold to determine the quality of cultured cells while it may be affected by any different culture conditions. Because the p1 cells/colony motion index is correlated with epithelial regenerative capacity, it is a reliable index for quality control of oral keratinocytes.

A Preliminary Study of Constructing Tissue Engineered Oral Mucosa

Adipose derived stem cells (ADSCs) have a great potential for tissue-engineering purposes, and they may be introduced in oral mucosa tissue engineering for urethroplasty. This study was aimed to develop a tissue-engineered oral mucosa through seeding oral keratinocytes (OKs) and adipose derived stem cells (ADSCs) on small intestine submucosa (SIS). From August 2018 to October 2019, an observational study was conducted in the laboratory of our hospital, to develop a tissue-engineered oral mucosa.SIS was obtained from porcine small intestine, and OKs and ADSCs were obtained from canine sources and were cultured and expanded in vitro. The two cell lines were seeded on the two surfaces of the SIS, and the cell-scaffold compound graft was cultured in an air fluid level for 1 week. The SIS exhibited a porous membrane, and no cells were found through HE staining. The model cultured with OK-SIS only formed a thin and loose epithelium. Whereas the model cultured with OK-SIS-ADSC was much thicker and denser. The co-cultured of ADSC and OK grew well on the SIS in which the OKs formed a multilayer of epithelium. So it is feasible to construct a tissue-engineered oral mucosa graft with ADSCs, OKs and SIS. The ADSCs contribute to a thicker epithelium formation.

Manufacturing micropatterned collagen scaffolds with chemical-crosslinking for development of biomimetic tissue-engineered oral mucosa

The junction between the epithelium and the underlying connective tissue undulates, constituting of rete ridges, which lack currently available soft tissue constructs. In this study, using a micro electro mechanical systems process and soft lithography, fifteen negative molds, with different dimensions and aspect ratios in grid- and pillar-type configurations, were designed and fabricated to create three-dimensional micropatterns and replicated onto fish-scale type I collagen scaffolds treated with chemical crosslinking. Image analyses showed the micropatterns were well-transferred onto the scaffold surfaces, showing the versatility of our manufacturing system. With the help of rheological test, the collagen scaffold manufactured in this study was confirmed to be an ideal gel and have visco-elastic features. As compared with our previous study, its mechanical and handling properties were improved by chemical cross-linking, which is beneficial for grafting and suturing into the complex structures of oral cavity. Histologic evaluation of a tissue-engineered oral mucosa showed the topographical microstructures of grid-type were well-preserved, rather than pillar-type, a well-stratified epithelial layer was regenerated on all scaffolds and the epithelial rete ridge-like structure was developed. As this three-dimensional microstructure is valuable for maintaining epithelial integrity, our micropatterned collagen scaffolds can be used not only intraorally but extraorally as a graft material for human use.

Tissue Engineering of Oral Mucosa and Salivary Gland: Disease Modeling and Clinical Applications

Oral mucosa and salivary gland are composed of complex and dynamic networks of extracellular matrix, multiple cell types, vasculature, and various biochemical agents. Two-dimensional (2D) cell culture is commonly used in testing new drugs and experimental therapies. However, 2D cell culture cannot fully replicate the architecture, physiological, and pathological microenvironment of living human oral mucosa and salivary glands. Recent microengineering techniques offer state of the science cell culture models that can recapitulate human organ structures and functions. This narrative review describes emerging in vitro models of oral and salivary gland tissue such as 3D cell culture models, spheroid and organoid models, tissue-on-a-chip, and functional decellularized scaffolds. Clinical applications of these models are also discussed in this review.

Influence of magnetized abutment on biological seal of the implant–soft tissue interface by reconstructed human gingiva (RHG) model

Background: osseointegration in implants have received a lot of attention. Recently, the focus has extended towards the development of abutment materials supporting gingival soft tissue attachment. The attachment of the soft tissue to the implant/abutment surface is necessary to form a biological seal that protects the pathogenic microbial colonization which can lead to periimplantitis and bone resorption culminating in dental implant failure. Aim/Hypothesis: To evaluate the Influence of magnetized abutment surface on biological seal of the implant–soft tissue interface that would enable junctional epithelium(JE) attachment to the abutment through an internal basal lamina and hemidesmosomes in order to prevent the JE from migrating too in depth. Materials and Methods: We developed novel magnetic nanoparticles (MNs) coated with chitosan(CS) (CS-MNs) for enhancing cellular migration using magnetic force. CS-MNs were characterized by XRD, UV, SEM. The CS-MNs where spherical shape with a diameter from 10 nm to 15 nm. The magnetized abutments surface was treated with CS-MNs. Influence of CS-MNs treated abutment on reconstructed human gingiva (RHG) model consisting of differentiated gingival epithelium cells was assessed in terms of Epithelial attachment, down-growth along the abutment surface, quantitative analysis by permeability and cell attachment tests. Phenotype via histomorphology, scanning electron microscopy, and immunohistochemistry 10 days after implantation. Results: Abutment values obtained from the intraexperiment replicates including measurement of the left and right side of the abutment (total of four measurements) were first averaged, and Differences were determined using one-way ANOVA followed by Dunnett's uncorrected multiple comparisons test. Differences were considered significant at P < 0.05. histomorphometric analyses showed epithelial growth near perpendicular to the surface of abutments. The magnification clearly showed individual keratinocytes spread and attached to abutment surfaces via filopodia extensions. Conclusions and Clinical Implications: The current RHG model shows the active migration of epithelial cells near perpendicularly along the magnetized abutment surface with higher gingival fibroblast attachment as compared with an untreated surface. This novel system is thought to be useful in the development of biological seal around the implant and cell-based strategies for the repair or replacement of tissue and other novel therapies. Keywords: Soft tissue-implant interactions, biological seal, tissue-engineered oral mucosa, three-dimensional in vitro model

Fibroblast encapsulation in gelatin methacryloyl (GelMA) versus collagen hydrogel as substrates for oral mucosa tissue engineering

PurposeOver the past decades, a variety of biomaterials have been investigated in terms of their suitability for oral mucosa tissue engineering. The aim of this study was to compare collagen and GelMA hydrogels as connective tissue scaffolds for fibroblasts and as substrates for seeding and culture of oral epithelial keratinocyte cells. MethodsHuman primary oral fibroblast and keratinocyte cells were isolated from gingival biopsies. The mixture of fibroblasts with GelMA or collagen gel were aliquoted within six-well tissue culture plate inserts and cross-linked using visible light or reconstitution buffer/heat, respectively. The viability of fibroblasts in the hydrogels was investigated after one and three days of cultivation using the PrestoBlue assay. Following the addition and culture of oral keratinocytes onto the connective tissue constructs, the tissue-engineered oral mucosa was assessed histologically. ResultsThe tissue viability assay shows that collagen hydrogels encapsulating fibroblasts displayed significantly higher cell viability than cell-laden GelMA constructs after 24 and 72 h (p < 0.05). A stratified and differentiated epithelium has formed on the surface of cell-laden collagen hydrogel but not on the surface of the GelMA-based substrate. ConclusionCollagen-based scaffold offers superior biological properties compared to GelMA hydrogel in terms of oral fibroblast growth, as well as epithelial cell adhesion and differentiation. Therefore, collagen-based hydrogels remain the preferred choice for oral mucosa tissue engineering.

In Vitro Degradation of Electrospun Poly(Lactic-Co-Glycolic Acid) (PLGA) for Oral Mucosa Regeneration.

Poly(lactic-co-glycolic acid) (PLGA) has been used in the field of tissue engineering as a scaffold due to its good biocompatibility, biodegradability and mechanical strength. With the aim to explore the degradability of PLGA electrospun nonwoven structures for oral mucosa tissue engineering applications, non-irradiated and gamma irradiated nonwovens were immersed in three different solutions, in which simulated body fluid (SBF) and artificial saliva are important for future oral mucosa tissue engineering. The nonwovens were immersed for 7, 15 and 30 days in SBF, culture media (DMEM) and artificial saliva at 37 °C. Before immersion in the solutions, the dosage of 15 kGy was applied for sterilization in one assay and compared with non-irradiated samples at the same timepoints. Samples were characterized using different techniques such as scanning electron microscopy (SEM), differential scanning calorimetric (DSC) and gel permeation chromatography (GPC) to evaluate the nonwoven degradation and Fourier-transform infrared spectroscopy (FTIR) to evaluate the chain scissions. Our results showed that PLGA nonwovens were constituted by semicrystalline fibers with moderate degradation properties up to thirty days. The non-irradiated samples exhibited slower kinetics of degradation than irradiated nonwovens. For immersion times longer than 7 days in the three different solutions, the mean diameter of irradiated fibers stayed in the same range, but significantly different from the control sample. On non-irradiated samples, the degradation kinetics was slower and the plateau in the diameter value was only attained after 30 days of immersion in the fluids. Plasticization (fluid absorption into the fiber structure) occurred in the bulk material, as confirmed by a decrease in Tg observed by DSC analyses of non-irradiated and irradiated nonwovens, in comparison with the respective controls. In addition, artificial saliva showed a higher capacity of influencing PLGA crystallization than SBF and DMEM. FTIR analyses showed typical PLGA chemical functional groups changes. These results will be important for future application of those PLGA electrospun nonwovens for oral mucosa regeneration.

Implant Soft-Tissue Attachment Using 3D Oral Mucosal Models-A Pilot Study.

Purpose: The aim of this study was to investigate soft-tissue attachment to different metal, ceramic, and polymer implant surfaces using an inflamed, three-dimensional (3D), tissue-engineered, human oral mucosal model, as well as multiple-endpoint qualitative and quantitative biological approaches. Methods: Normal human oral fibroblasts, OKF6/TERT-2 keratinocytes and THP-1 monocytes were cultured, and full-thickness, 3D oral mucosal models were engineered inside tissue culture inserts. Sand-blasted and acid-etched (SLA) and machined (M) titanium–zirconium alloy (TiZr; commercially known as Roxolid; Institut Straumann AG, Switzerland), ceramic (ZrO2), and polyether ether ketone (PEEK) rods (Ø 4 mm × 8 mm) were inserted into the center of tissue-engineered oral mucosa following a Ø 4mm punch biopsy. Inflammation was simulated with addition of the lipopolysaccharide (LPS) of Escherichia coli (E. coli) and tumor necrosis factor (TNF)-alpha to the culture medium. Implant soft-tissue attachment was assessed using histology, an implant pull-test with PrestoBlue assay, and scanning electron microscopy (SEM). Results: Inflamed, full-thickness, 3D human oral mucosal models with inserted implants were successfully engineered and histologically characterized. The implant pull-test with PrestoBlue assay showed higher viability of the tissue that remained attached to the TiZr-SLA surface compared to the other test groups. This difference was statistically significant (p < 0.05). SEM analysis showed evidence of epithelial cell attachment on different implant surfaces. Conclusions: The inflamed, 3D, oral mucosal model has the potential to be used as a suitable in vitro test system for visualization and quantification of implant soft-tissue attachment. The results of our study indicate greater soft tissue attachment to TiZr-SLA compared to TiZr-M, ceramic, and PEEK surfaces.

195 - Mid-term results of anterior urethroplasty with tissue-engineered oral mucosa graft MukoCell®

195 - Mid-term results of anterior urethroplasty with tissue-engineered oral mucosa graft MukoCell®

MP60-12 MID-TERM RESULTS OF ANTERIOR URETHROPLASTY WITH TISSUE-ENGINEERED ORAL MUCOSA GRAFT MUKOCELL®

INTRODUCTION AND OBJECTIVE: Anterior urethroplasty with buccal mucosa grafting is the standard procedure for recurrent urethral strictures of ≥2 cm length. Trying to avoid oral complications through harvesting buccal mucosa, a tissue-engineered oral mucosa graft (MukoCell®) was manufactured. Current multicenter retrospective studies report success rates of 67-84%. The objective of our institutional retrospective analysis is the assessment of the mid-term efficacy and complications of this method. METHODS: Between 05/2016 and 06/2019 a total of 77 patients with anterior urethral strictures underwent a urethroplasty with MukoCell®. Our cohort consisted of 45 patients with bulbar strictures (78,9%), 15 with penobulbar (19,5%), 11 with penile (14,3%), 4 with membranous (5,2%) and 2 with simultaneous bulbar and penile strictures (2,6%) respectively. Mean patient age was 59 years (range 16 to 87) with mean 2,5 previous endoscopic or anastomotic procedures (range 0 to 8). 4 different surgeons performed in 61 cases ventral/dorsal onlay techniques (79,2%), in 5 cases inlay-ASOPA technique (6,5%) and in 11 cases combined (Palminteri et al.) technique (14,3%). Mean stricture length was 5,3 cm (range 2 to 16). 8 (10,4%) strictures were traumatic, 39 (50,6%) iatrogenic, 21 (27,3%) idiopathic, 1 (1,3%) radiogenic, 1 (1,3%) associated with Lichen sclerosus, 2 (2,6%) former hypospadias and 2 (2,6%) had other causes. IPSS-Score, Uroflowmetry and post void residual measurement were recorded pre- and postoperatively twice a year. Retrograde urethrography was performed pre- and 3 weeks postoperatively and repeated by suspicion of recurrence. RESULTS: Mean follow up was 24 months (range 8 to 43). 24 patients (31,2%) developed a recurrence of the stricture (14 with bulbar, 4 penobulbar, 3 penile, 2 membranous and 1 simultaneous bulbar and penile localization and mean 2,3 previous endoscopic operations (range 1-6) after mean 6,5 months (range 1,5 to 17). None perioperative hemorrhage occurred in all patients. We reported 5 cases of perineal wound dehiscence, 5 UTIs, 7 contrast leaks in the postoperative urethrography and 1 urethrocutaneous fistula requiring surgical intervention (Clavien-dindo 3b). No local (oral-urethral) or general adverse events related to the use of MukoCell® were observed. CONCLUSIONS: The recurrence-free rate of anterior urethroplasty using MukoCell® in our hands was 68,8% after a two-year follow-up. A prospective multicenter study comparing „head-to-head“ the efficiency of this graft with the native oral mucosa will be initiated in Germany in 2020. Source of Funding: none

Development of microstructured fish scale collagen scaffolds to manufacture a tissue-engineered oral mucosa equivalent

The present study aimed to develop a more biomimetic tissue-engineered oral mucosa equivalent comprising 1% type I tilapia scale collagen scaffold having microstructures mimicking the dermal–epidermal junction of oral mucosa and oral keratinocytes as graft materials for human use. We designed four micropattern prototypes mimicking the dermal–epidermal junction. Using a semiconductor process and soft lithography, negative molds were fabricated to develop microstructures using both polydimethylsiloxane and silicon substrates. Micropattern configurations of dermal–epidermal junctions manufactured from fish collagen consisting of a fibril network using our micropatterning system were well preserved, although the internal fibril network of the pillar pattern was sparse. Mixing 1% chondroitin sulfate with the collagen matrix minimized tissue-engineered oral mucosa equivalent contraction. Histologic examinations showed a flattening of the vertical dimensions of all microstructures and expansion of their pitches, indicating changes in the originally designed configurations. Nonetheless, histologic examinations revealed that a fully differentiated and stratified epithelial layer was developed on all scaffolds, suggesting that the microstructured fish scale collagen scaffolds have potential in the manufacturing of tissue-engineered oral mucosa equivalents for clinical use; however, enhancement of the mechanical properties of micropatterns is required. Our micropatterning technology can also apply to the development of oral mucosa in vitro models.

Tissue engineering of the urethra: where are we in 2019?

PurposeThe purpose of this review is to assess the potential role of tissue engineering for urethral reconstruction. It is well- recognised that urethrotomy remains the first-line therapy in the treatment of urethral stricture. Following on from the randomised study which recommended no difference between urethrotomy and urethral dilation, Steenkamp et al. reported long-term success rates of only 20%. Patients with longer strictures, penile or distal urethral strictures, and extensive periurethral spongiofibrosis typically do not respond well to repeated incisions. This report reviews the potential role of tissue engineering as applied to augmentation urethroplasty, which is the treatment of choice following failed urethrotomy.MethodsA review of the literature was carried out. The principal emphasis was on tissue engineering as applied to augmentation urethroplasty, but an introductory section reviews the use of urethrotomy and the background to contemporary practise with augmentation urethroplasty using oral mucosa.ResultsIt is evident that a cellular matrix which requires the ingrowth of cells is unlikely to be successful except for very short strictures. Other approaches such as injection of stem cells have not been adequately trialled in humans to date. Tissue-engineered substitute for autologous oral mucosa has been used and the results relating to this are reviewed.ConclusionsTissue engineering of autologous tissue for urethroplasty is expensive. It is unnecessary for the majority of cases, but could be potentially useful for very lengthy strictures, for instance, relating to lichen sclerosis. Whilst tissue-engineered oral mucosa has been successfully used, a great deal more work would be necessary to develop an appropriate matrix. Another study has looked at a larger series using an alternative tissue-engineered substitute, but the results have been very disappointing. At present, it has to be concluded that there is no effective and validated tissue engineering solution for the management of urethral stricture disease.

PT016 - Augmentation urethral reconstuction using tissue-engineered oral mucosa graft MukoCell®

PT016 - Augmentation urethral reconstuction using tissue-engineered oral mucosa graft MukoCell®

Pre-clinical evaluation of novel mucoadhesive bilayer patches for local delivery of clobetasol-17-propionate to the oral mucosa

Oral lichen planus (OLP) and recurrent aphthous stomatitis (RAS) are chronic inflammatory conditions often characterised by erosive and/or painful oral lesions that have a considerable impact on quality of life. Current treatment often necessitates the use of steroids in the form of mouthwashes, creams or ointments, but these are often ineffective due to inadequate drug contact times with the lesion. Here we evaluate the performance of novel mucoadhesive patches for targeted drug delivery. Electrospun polymeric mucoadhesive patches were produced and characterised for their physical properties and cytotoxicity before evaluation of residence time and acceptability in a human feasibility study. Clobetasol-17-propionate incorporated into the patches was released in a sustained manner in both tissue-engineered oral mucosa and ex vivo porcine mucosa. Clobetasol-17 propionate-loaded patches were further evaluated for residence time and drug release in an in vivo animal model and demonstrated prolonged adhesion and drug release at therapeutic-relevant doses and time points. These data show that electrospun patches are adherent to mucosal tissue without causing tissue damage, and can be successfully loaded with and release clinically active drugs. These patches hold great promise for the treatment of oral conditions such as OLP and RAS, and potentially many other oral lesions.

Tissue-engineered autologous oral mucosa graft for urethral reconstruction: Results from a national multicentre survey

Tissue-engineered autologous oral mucosa graft for urethral reconstruction: Results from a national multicentre survey

V29 - Urethral reconstruction using tissue-engineered oral mucosa graft MukoCell® in onlay technique

V29 - Urethral reconstruction using tissue-engineered oral mucosa graft MukoCell® in onlay technique