Research publications

Introduction: The causes of tooth loss are very diverse, including trauma, tumor removal, tooth decay, and—very commonly—periodontitis (bacterial inflammation of the gums and jawbone). In Germany, approximately 50–60% of the population suffers from moderate to severe periodontitis, with the incidence increasing with age; consequently, a growing demand for artificial teeth is expected in the future [1]. In addition to the root cavity resulting from tooth loss, the causes mentioned above can also lead to damage to larger areas of the jawbone or necessitate their removal. Furthermore, bone resorption occurs due to the lack of mechanical stress on the jaw, making it impossible to place an implant without prior reconstruction of the bone tissue. Since an implant must be firmly anchored in the jawbone, prior bone augmentation is therefore absolutely necessary in these cases to provide patients with optimal care. In Germany, over 50% of dental implants placed require such augmentation [2–4]. In this treatment approach, a bone substitute material is filled into the cavity and covered with a GBR membrane. The membrane is essential for the success of the treatment, as its primary function is to prevent rapidly proliferating soft tissue cells from invading the cavity. Currently, both non-resorbable (e.g., PTFE, titanium) and resorbable (e.g., collagen) GBR membranes are used; however, they can only partially meet the required performance criteria. Resorbable membranes tend to be preferred because they do not need to be removed in a second surgical procedure—which could jeopardize treatment success—and carry a lower risk of infection. However, due to their lack of primary stability, their mechanical properties do not yet allow them to maintain space in a moist environment. For this reason, non-resorbable GBR membranes continue to be used as well [5, 6]. A GBR membrane that combines resorbability with space-maintaining properties in a way that meets clinical requirements does not yet exist, a fact underscored by high revision rates of approximately 20% [7, 8].

The IGF OsteoMe project aimed to develop novel, multilayer GBR membranes for bone augmentation with a complex, application-oriented property profile. To this end, the project partners ITM and FILK combined their complementary expertise in the development of electrospun and additive-manufactured (Fiber Additive Manufacturing, FAM) biopolymer structures, the functionalization of chitosans, and cell biological characterization. The property profile, which had been developed in advance through discussions with clinicians and industry representatives, was to be realized through a three-layer membrane structure consisting of a mucoadhesive layer for positional stabilization, a cell-excluding barrier layer, and a structuring, mineralized layer with defined porosity and mineral gradients to biomimetically replicate the bone-tissue interface and provide space-maintaining properties. Based on the materials polycaprolactone (PCL), silk fibroin (SF), and chitosan (Ch), simulation-based fabrication strategies were investigated that enable the targeted adjustment of mechanical, functional, and biological properties.

Results:

Mucoadhesive layer: Mucoadhesive materials allow for prolonged retention on mucous membranes and are therefore of interest for numerous medical applications. Chitosan is considered a promising mucoadhesive biopolymer due to its positive charge and the resulting interactions with negatively charged mucins. To further improve adhesion, the covalent binding of thiol-containing compounds was investigated, as these additionally enable the formation of disulfide bonds with mucin.

Functionalization was carried out via carbodiimide (EDC/NHS)-mediated coupling of N-acetylcysteine (NAC) or cysteine to chitosan with a degree of deacetylation exceeding 90%. Both the direct modification of chitosan films and the derivatization of dissolved chitosan were investigated. While the functionalization of films was complicated by the limited accessibility of the amino groups within the solid polymer matrix and resulted in an inhomogeneous distribution of thiol groups, the modification of dissolved chitosan enabled the preparation of reproducible conjugates with high thiol contents. The optimized chitosan conjugate achieved a total thiol content of approximately 878 µmol/g, with about 311 µmol/g present as free thiol groups. Due to the high reactivity of the thiol groups, a loss of approximately 20% was observed within two weeks during storage in air; therefore, storage under oxygen-free conditions is recommended. To produce mechanically stable membranes, the addition of at least 70 wt% unmodified chitosan was required (see Figure 1).

The mucoadhesive properties were investigated using a specially developed test setup on a texture analyzer, with porcine intestinal mucosa serving as the model substrate. The adhesive forces were determined relative to unmodified chitosan. In particular, acid-treated chitosan films exhibited high adhesion forces, which could be specifically influenced by varying the contact pressure and contact time. Neutralized films, on the other hand, did not achieve the target adhesion forces. Even the introduction of thiol groups did not lead to a significant improvement in mucoadhesion under neutral conditions (see Figures 2 and 3).

Although the formation of disulfide bonds between thiol groups and mucin is considered an established mechanism for enhancing mucoadhesion, this effect could not be demonstrated in the present studies. Possible causes under discussion include an insufficient density of reactive thiol groups on the surface and their limited reactivity under physiological conditions.

In summary, it was demonstrated that thiol-modified chitosan materials can be successfully produced. However, the highest adhesion forces were achieved not through chemical functionalization, but through acid-treated chitosan films. For future applications, strategies for stabilizing and controllably neutralizing these highly adhesive materials therefore represent a particularly promising avenue for development.

Barrier layer: To create submicroscale pores, the interactions between process and spinning solution parameters as well as fiber diameters were first investigated in detail. The spinning solution concentration (2–15%), the silk fibroin (SF)–polycaprolactone (PCL) ratio (0–100% SF), the influence of salt additives (up to 5 wt-% potassium chloride - KCl), the flow rate (0.5–2 ml/h), and the electric field strength (0–1.5 kV/cm) were systematically varied. It was found that fiber diameters can be specifically reduced, in particular by lowering the flow rate and the spinning solution concentration (Figure 4A) as well as by adding up to 66% SF. Higher SF content and salt additives lead to inhomogeneities in the resulting fiber diameters and shapes, as well as, in some cases, to embrittlement of the membranes. Varying the electric field strength (0–40 kV) also affected the fiber diameters, though only to a very small extent (approx. ± 22 nm); therefore, the preferred solution was selected here based on differences in handling during membrane production. Concentrations below 3% were not spinnable or resulted in irregular strand breaks. Based on these findings, the case group SF:PCL_1:2_3% was selected as the preferred solution. This case group exhibited nearly normally distributed pore sizes with a mean of 613 nm (Figure 4B). Approximately 6% of the pores had a diameter below the target value of 200 nm. To investigate migration behavior, the membranes were mounted in cell culture inserts (Cell-Crown™ inserts) and seeded with gingival epithelial cells for 72 h. After cultivation, cryosections were prepared, and the cytoskeleton and cell nuclei were stained. The barrier function was demonstrated over a period of 72 h (Figure 4C).

Mineral Layer: To achieve the desired structure of macroporous reinforcement structures, a tricalcium phosphate (TCP)-containing printing paste based on PCL was developed for the FAM. Mineral content of up to 50% was achieved without compromising printability. Using the FAM, constructs with triangular pore geometries were produced, as these offered advantages in terms of surgical handling, particularly with regard to flexibility. The 3D-printed specimens were subjected to enzymatic-catalyzed degradation over an 8-week period and, depending on the degree of degradation, were mechanically characterized in a humid environment and compared with commercially available, non-degraded GBR membranes (BioGuide, CollProtect, Mucoderm). To this end, four test specimens per week were incubated for 8 weeks in a degradation solution (physiological phosphate-buffered saline (PBS) and an enzyme cocktail consisting of protease XIV and lipase). The medium was changed twice each week to prevent the degradation solution from becoming saturated with degradation products. The enzyme concentration was set at 2.5 U/L per enzyme so that both the lipase and protease, considered individually, would reflect the total enzyme concentration in human saliva [9]. This increased enzyme concentration enables rapid degradation while also providing reliable data on the minimum shelf life of the developed GBR membrane in the human oral cavity. Initially, a slight increase in mass and tensile strength in the wet state was observed, which is presumably due to the deposition of salts on the surface (Figure 5). Starting in week 3, a continuous decline in mass and mechanical properties can be observed, although this decline is comparatively minor. Even after 7 weeks, the tensile strength remains in the range of 8 MPa and is thus comparable to the initial strength prior to degradation. Thus, the space-maintaining capacity of the developed GBR membrane was demonstrated for approximately 2 months, which is sufficient for the remodeling of natural bone tissue. The target value of > 5 MPa in a moist environment was maintained throughout the entire degradation period. Furthermore, a comparison was conducted with non-degrading, commercially available GBR membranes, demonstrating the comparability of the developed membrane. In addition, continuous calcium release was detected over the course of the degradation period, which may have a positive effect on bone regeneration.

To evaluate the osteoconductive effect of the mineral layer, the differentiation of bone progenitor cells (mesenchymal stem cells, MSCs) into osteogenic cells was investigated. Based on the staining of calcium deposits in the mineralized tissue (Alizarin Red S staining) and the quantitative analysis of osteogenic marker expression (qPCR of collagen I (Col1), alkaline phosphatase (ALP), osteocalcin (BGLAP), and osteopontin (SPP1)), a positive effect of the membrane developed in the project on bone tissue regeneration was demonstrated. Osteogenic induction was achieved by adding dexamethasone, β-glycerophosphate, and ascorbic acid for 28 days. Differentiation was controlled using MSCs without any material influence.

Summary: As a result of the OsteoMe project, three membrane layers were developed, each of which fulfills essential aspects of the requirements profile for GBR membranes in dentistry. Depending on the acidity of the chitosan as well as the contact time and pressure, the mucoadhesive layer develops adhesive forces in the range of 1 to 4 N, which are comparable to those of established fibrin glues. This enables the membrane to be applied to the defect site in a position-stable manner without the need for additional sutures or pins. The barrier layer is designed with open pores, allowing for the exchange of nutrients and signaling molecules, which can positively influence regeneration. At the same time, it was shown that the pore size is small enough to act as a barrier to rapidly proliferating oral mucosal cells, thereby providing the slow-growing bone tissue cells with sufficient time for regeneration. The 3D-printed mineral layer exhibits sufficiently high mechanical properties in simulated saliva to ensure a space-maintaining function. The tensile strengths achieved are comparable to those of commercially available GBR membranes and show virtually no decline during enzymatic-catalyzed degradation over eight weeks. Furthermore, it was demonstrated that the mineral layer exhibits osteoinductive properties and can thus actively support bone regeneration. Finally, methods were developed to ensure a delamination-free bonding of the individual layers. This allows the layers to be flexibly combined with one another, thereby expanding the range of possible applications.

 

Acknowledgments: The IGF project 01IF22810N of the Forschungskuratorium Textil e.V. research association was funded by the Federal Ministry for Economic Affairs and Energy through the DLR Project Management Agency as part of the Program for the Promotion of Industrial Collaborative Research (IGF), pursuant to a resolution of the German Bundestag.

         

Quellenverzeichnis:

[1]         CHOLMAKOW-BODECHTEL, Constanze: Fünfte Deutsche Mundgesundheitsstudie (DMS V). JORDAN, Andreas Rainer (Hrsg.); MICHEELIS, Wolfgang (Hrsg.). Köln : Deutscher Zahnärzte Verlag DÄV, 2016

[2]         CHA, Hyun-Suk ; KIM, Ji-Wan ; HWANG, Jong-Hyun ; AHN, Kang-Min: Frequency of bone graft in implant surgery. In: Maxillofacial plastic and reconstructive surgery 38 (2016), Nr. 1, S. 19

[3]         MOY, Peter K. ; AGHALOO, Tara: Risk factors in bone augmentation procedures. In: Periodon-tology 2000 81 (2019), Nr. 1, S. 76–90

[4]         KNÖFLER, Wolfram ; BARTH, Thomas ; GRAUL, Reinhard ; KRAMPE, Dietmar: Retrospective analysis of 10,000 implants from insertion up to 20 years-analysis of implantations using aug-mentative procedures. In: International journal of implant dentistry 2 (2016), Nr. 1, S. 25

[5]         CABALLÉ-SERRANO, Jordi ; MUNAR-FRAU, Antonio ; ORTIZ-PUIGPELAT, Octavi ; SOTO-PENALOZA, David ; PEÑARROCHA, Miguel ; HERNÁNDEZ-ALFARO, Federico: On the search of the ideal barrier membrane for guided bone regeneration. In: Journal of clinical and experi-mental dentistry 10 (2018), Nr. 5, e477-e483

[6]         JIMÉNEZ GARCIA, J. ; BERGHEZAN, S. ; CARAMÊS, J. M. M. ; DARD, M. M. ; MARQUES, D. N. S.: Effect of cross-linked vs non-cross-linked collagen membranes on bone: A systematic re-view. In: Journal of periodontal research 52 (2017), Nr. 6, S. 955–964

[7]         BUTENSCHÖN, Sina: Prävalenz periimplantärer Entzündungen bei teilbezahnten Patienten nach einer minimalen Beobachtungsdauer von 10 Jahren - eine retrospektive Querschnittsstu-die. Göttingen, Georg-August-Universität zu Göttingen. Dissertation. 2019

[8]         RAKIC, Mia ; GALINDO-MORENO, Pablo ; MONJE, Alberto ; RADOVANOVIC, Sandro ; WANG, Hom-Lay ; COCHRAN, David ; SCULEAN, Anton ; CANULLO, Luigi: How frequent does peri-implantitis occur? A systematic review and meta-analysis. In: Clinical oral investigations 22 (2018), Nr. 4, S. 1805–1816

[9]         CHAUNCEY, Howard Haskell. The chemical composition of human sal

Initial situation and problem definition

The windpipe (trachea) fulfils two main functions: (I) it provides an airtight and mechanically stable passage from the larynx to the bronchial tree of the lungs for air transport, and (II) it facilitates the transport of mucus. Function (I) is performed by a tubular structure consisting of cartilage rings and longitudinal muscles, which provide lateral stability and longitudinal flexibility. This keeps the lumen open for breathing air. In addition, the inhaled air is moistened and warmed. Function (II) is a cleaning mechanism that is performed by a special mucous membrane layer (mucociliary respiratory epithelial layer). Here, mucus-producing cells and cells with tiny hairs (cilia) on their surface transport mucus and particles [2].

After an injury to the trachea, these functions are impaired by the insertion of a tracheal cannula. In Germany, 53,000 tracheal resections (replacement of part of the trachea) are performed annually [3]. A high proportion, around 40,000 patients, receive non-clinical care through a surgically created opening in the windpipe, known as a stoma [3]. This treatment has significant disadvantages: 1. poorer humidification and warming of the inhaled air, 2. poorer sense of smell and taste, 3. impaired voice formation, and 4. narrowing of the windpipe.

The gold standard for tracheal reconstruction is end-to-end anastomosis, in which part of the trachea is removed and the remaining ends are sutured together [4]. However, for this procedure to be performed, at least half of the trachea must remain in adults and one third in children, otherwise the operation cannot be carried out [5]. Nevertheless, complications occur in up to 20% of cases [6]. As the sutured ends of the trachea are subject to considerable force, this can lead to the suture tearing and the trachea shifting into the chest cavity. There is also a risk that the ends will not grow together properly, leading to scarred narrowing of the trachea, tracheitis, hoarseness, loss of voice and paralysis of the vocal cord nerves, as well as swallowing disorders [7]. Approaches investigated to date – including synthetic implants, constructs made from the patient's own tissue, donor tracheas and tissue engineering procedures – have not yet been able to replicate a functional cilia layer for mucus and particle transport. Neither this lack of transport function nor the high complication rate and shortage of suitable donor tissue currently allow for reliable use in cases of larger tracheal defects following clinically necessary resection. As a result, there is currently no implant available that can adequately replace the trachea.

It is therefore necessary to develop novel implants that mimic both the mechanical stability and the internal transport function of the natural trachea. The aim of the IGF project was therefore to develop a textile, functional and biomimetic tubular fabric. This fabric should have a cilia-like structure for active substance transport. At the same time, rigid, 3D-printed support structures, which can be integrated during the weaving process, were to protect the tubular fabric from collapsing. Both aspects serve to safely bridge missing or removed tracheal tissue. The ciliary movement should be achieved by electroactive piezoelectric PVDF fibres integrated into the tissue in the form of polarised naps. The ciliary movement is to be activated by the piezoelectric effect, which is triggered by the electric field generated by current-carrying conductors.

Development of tubular tissue structures

To produce a tubular fabric with cilia on the fabric surface, various variants were developed for a multi-layer fabric with naps pointing into the interior of the tube. The fabrics were manufactured using commercially available shuttle loom technology with a Jacquard unit for versatile adaptation of the fabric structure.

The tubular base structure was woven from polyester threads. Depending on the variant, cilia threads or a combination of cilia threads (piezoelectric PVDF or Nitinol threads) and conductor threads (silver-plated polyamide, Madeira HC40) were incorporated into the base fabric. The use of conductor threads was necessary when using electroactive PVDF multifilament threads or short fibres to stimulate cilia movement. When using one-way or two-way shape memory (SM) filaments as cilia material, no separate conductor filaments had to be incorporated into the fabric, as the SM filaments were directly contacted and conductive in order to initiate the movement of the cilia.

Development of biomimetic support structures

The human trachea has approximately 15 to 20 tracheal cartilages. They are horseshoe-shaped, have a diameter of 20 mm, with the open side facing dorsally (towards the back), and are approximately 4 mm wide and 1 mm thick. Their outer surface is flat and the inner surface is convex. Tracheal cartilages that can be integrated into the web (cartilage clips/support structures) should be manufactured using 3D printing and should be able to withstand a compression force of at least 1.2 N.

Based on this geometry, a total of 10 different models were developed. The differences in geometry resulted from variations in the leg geometry (C- and U-shaped), wall thickness and radius. The support structures were produced using photopolymer printing based on the stereolithography concept with an Objet 30 Prime from Stratasys in order to achieve the necessary geometric details. Exemplary structures are shown in Figure 1.

To examine the cartilage structures, clamps that meet the requirements for commercially available measurement technology were developed, designed and implemented using 3D printing. The clamps developed enable pressure loading in various anatomical positions of the cartilage segments (anterior-posterior & medial-lateral).

Integration of support structures into the tissue structure

Based on the previously presented woven tubular fabric, including the naps anchored in the base fabric on the fabric surface, a weaving structure was developed that could accommodate and fix the developed support structures at defined intervals in the base fabric. The integration of the support structures was achieved by weaving a fabric pocket over the entire circumference of the fabric. The dimensions (width and thickness) of the fabric pocket were adapted to those of the support structures, which were fixed between two layers of fabric and secured against slipping and "twisting out" of the structure. The number of support structures per defined fabric length was adjustable in terms of binding, and different clip widths could also be integrated into the tubular fabric by adjusting the fabric pocket size. The implemented demonstrator is shown in Figure 3. The inward-facing cilia and tissue pockets with the integrated support structures are clearly visible.

Textile physical analysis of the support and tissue structures as well as movement analysis

The average tensile strength of the human trachea is approximately 230 N [8]. The tubular tissue structures with integrated support structures exhibited a maximum tensile strength of approximately 4300 N. A yield strength of approximately 1400 N was determined. This means that the mechanical requirements of the human trachea are fully met. All support structures developed to prevent the trachea from collapsing exhibited a compression force greater than 1.2 N. In some cases, the target value was exceeded tenfold.

In addition, the influence of repeated or cyclic tensile loading on the position of the support structures integrated into the tissue was investigated. To this end, a load cycle test with 150 cycles was performed, in which a tensile load of up to a maximum force of 230 N (target value) was repeatedly applied, followed by relief to the initial position. A sample holder was developed and implemented for this purpose so that the tubular structure was loaded biomimetically across the entire cross-section. The results show that the support structures woven into the tissue pockets remained firmly fixed and did not "twist out" in the circumferential direction. The selected integration and fixation method thus ensures permanent positional stability under cyclic loading.

Motion analysis of the various patterns showed that PVDF fibres did not enable ciliary movement. However, the SM filaments with a two-way effect demonstrated repeatable ciliary movement. This approach can be used in the future to replicate the functioning of human cilia. As a further alternative approach, fabrics with parallel conductor threads were flocked with polyamide short fibres. Using an alternating electric field, intermittent cilia movement could also be simulated here.

Summary

A novel tracheal implant was developed at ITM that excellently replicates the macroscopic structure of the human trachea. The developed structure could be manufactured using commercially available shuttle weaving technology without any design modifications. To maintain a pressure-stable tubular structure, 3D-printed support structures were integrated into tissue pockets. Production can be carried out integrally and can be adapted to individual patients in terms of tissue length, support structure spacing, number of support structures and pressure stability. In addition, various concepts were investigated to replicate the microscopic structure in order to generate mass transport. The basis for this was the creation of polnop tissue and the use of piezoelectric PVDF fibres. It was found that PVDF nubs did not allow for movement on a microscopic scale. Ciliary movement was achieved using other actuator fibres such as nitinol fibres. Ciliary movement can also be achieved using flock fibres.

Acknowledgements

The project ‘Tubular tissues with rigid and flexible structural zones and mass transport for the biomimetic construction of the trachea (01IF22889N)’ is funded by the Federal Ministry for Economic Affairs and Energy as part of the ‘Industrial Collective Research (IGF)’ programme on the basis of a resolution passed by the German Bundestag.

References

[1]  Aleksanya, A.; Stoelben, E.: Laryngotracheal resection as an alternative to permanent tracheostomy. Pneumologie 73 (2019), No. 4, pp. 211–218. URLhttps://www.thieme-connect.com/products/ejournals/html/10.1055/a-0809-0232

[2]  Udelsma, Brooks; Mathisen, Douglas J.; Ott, Harald C.: A reassessment of tracheal substitutes—a systematic review. In: Annals of Cardiothoracic Surgery 7 (2018), No. 2, pp. 175–182. URLhttps://www.annalscts.com/article/view/16458/16661

[3]  BVMe d:  BVMed provides information on tracheotomy and laryngectomy care. URL https://www.bvmed.de/verband/presse/pressemeldungen/bvmed-informiert-ueber-tracheotomie-und-laryngektomie-versorgung. – Update date: 19 May 2016 – Review date: 15 October 2025

[4]  Canzan, F.; Aggazzotti Cavazza, E.; Mattioli, F.; Ghidini, A.; Bottero, S.; Presutti, L.: Step-by-Step Tracheal Resection with End-to-End Anastomosis. In: Ghidini, Angelo; Mattioli, Francesco; Bottero, Sergio; Presutti, Livio (eds.): Atlas of Airway Surgery :  ACham: Springer International Publishing, 2017, pp. 75–82

[5]  Weme, Richard D.; Detamore, Michael; Weatherly, Robert A.: Immunohistochemical characterisation of rabbit tracheal cartilages. In: Journal of Biomedical Science and Engineering 03 (2010), No. 10, pp. 1007–1013

[6]  Damian o, Giuseppe; Palumbo, Vincenzo Davide; Fazzotta, Salvatore; Curione, Francesco; Lo Monte, Giulia; Brucato, Valerio Maria Bartolo; Lo Monte, Attilio Ignazio: Current Strategies for Tracheal Replacement: A Review.  In: Life 11 (2021), No. 7, pp. 618. URLhttps://www.mdpi.com/2075-1729/11/7/618

[7]  Rettinge, Gerhard; Hosemann, Werner; Hüttenbrink, Karl-Bernd; Werner, Jochen Alfred: ENT Surgery : . 5th, completely revised edition. Stuttgart: Thieme, 2018

[8]  A. Berghau s: . In: Cardiac, Thoracic and Vascular Surgery 1987 (1987), Volume 1. URL https://epub.ub.uni-muenchen.de/6218/1/6218.pdf – Review date 2025-10-15