Tissue engineering for temporomandibular joint disc repair and regeneration: a methodological perspective

Abstract

This perspective aims to highlight some of the methodological issues in tissue engineering, using the temporomandibular joint (TMJ) disc as an example. Knowing that disorders affecting the TMJ are quiet common and that implants that mimic such a complex structure remain a paramount challenge, tissue engineering has engaged in some efforts to solve this issue. Furthermore, tissue engineering should be considered a multidisciplinary scientific field, applying a wide variety of methodologies, thus requiring multidisciplinary research teams, to provide suitable inputs for its development. From our perspective, it is possible to corroborate that significant improvements have been done for engineering the TMJ disc; however, in vivo trials are scarce suggesting that further research is mandatory to obtain results that may lead to translational medicine.

In Context

The temporomandibular joint (TMJ) is one of the profusely operational joints in the body, registering over 2,000 periods of motion frequency per day. Therefore, it is clear that disorders affecting this joint are quite common. Tissue engineering (TE) is a promising field regarding tissue repair and regeneration, for which choosing the right approach is a major concern for suitable outcomes. Although there has been a huge amount of work aiming to regenerate cartilage, there are different types of cartilage that require different types of approach. In fact, for the TMJ disc a variety of techniques have been described in the literature, but no single technique has proven to be universally successful in reducing ankyloses recurrence and reproducing TMJ function. This perspective highlights some of the methods used to overcome this problem, addressing some possible insights for further research.

• temporomandibular joint
• tissue engineering
• scaffolds
• additive manufacturing
• ankyloses

The human body has a well-known difficulty to heal or repair injured tissues and organs. Moreover, these recoveries are commonly slow, deficient, or even impossible, leading to their dysfunctionality and sometimes patient's death. Therefore, many patients need ‘new’ organs or tissues, which are mainly donated by deceased people or, less frequently, by living donors. Nevertheless, the number of available donors is much lower than the number of new patients in the waiting list, presenting an exponential increase between this difference. In the last decade, the number of people on waiting lists for organ transplant in the European Union increased from around 51,000 in 2004 to 56,000 in 2014, considering the actual 28 member states (1). Nowadays, vital organs transplantation is the main treatment for their end-stage failure. As the transplantation is not suitable for every type of tissue in the human body, TE and regenerative medicine (RM) approaches have been getting promising results to achieve proper solutions for these concerns (2).

From the early 1970s, countless techniques have emerged in order to repair, regenerate, or replace injured tissues and organs, particularly synthetic skin substitutes and products. However, only almost two decades later, in 1987, the term ‘tissue engineering’ was first used by the National Science Foundation, triggering a fast progress of these techniques all over the world. Following this, TE has been stated as a multidisciplinary field combining cells and suitable factors (either biochemical, such as growth factors, or physical, such as cyclic mechanical loading), as well as biomaterials and engineering principles, in order to create biological substitutes to repair, regenerate, or replace injured tissues and organs (3).

Traditionally, TE uses an artificial extracellular matrix (ECM) to support cell growth and to promote the formation of tissues and organs, either in vitro or in vivo. The first approach is based on cell maturation and/or differentiation in an artificial matrix outside the body, and only then it is implanted. The in vivo approach is based on the same principle, but the cell-ECM structure is placed in situ for its maturation (4). Besides these approaches, other methods have been used aiming tissue regeneration, such as cell delivery, closed-loop methods, or cell and organ printing (4–6). The first method is based on the cell delivery into the bloodstream, or into the injured tissue by injection or infusion (7). The second method involves cell microencapsulation systems, with a semipermeable membrane that allows diffusion of nutrients and excreted products, and the release of therapeutic agents (8). The third mentioned method is based on inkjet printing for positioning cells, aiming to mimic organs’ structures (5, 9, 10).

Procedures for tissue formation

ECM structures are expected to respond to several requirements from the biological (allow cellular interaction, as well as cell adhesion, proliferation, migration, and/or differentiation) and mechanical (mimicking the morphological structure, as well as its function) points of view, being used in the form of scaffolds or hydrogels. Scaffolds are intentionally modified materials, comprising an interconnected porous network in order to promote the necessary interactions for the formation of new functional tissues. Hydrogels are polymeric hydrophilic networks that can expand to its maximum volume by water adsorption. These hydrogels have been used by many research groups for cell encapsulation or as a support material for organ printing processes (10, 11). Moreover, it can be noticed an increasingly interest in combining the scaffolds and hydrogels to obtain tailored constructs. These constructs must be biocompatible, biodegradable, or bioresorbable at a rate similar to new tissue regeneration and have a three-dimensional (3D) structure with appropriate porosity and high interconnectivity; mechanically, they should have proper mechanical resistance for hard tissues or viscoelasticity for soft tissues and should be produced with surface finish suitable for their application. The production of such structures can be obtained through several different techniques, which are chosen according to: 1) the final application or tissue to be regenerated, 2) the material that is intendent to be used, and 3) the requirements that need to be fulfilled to regenerate the damaged tissue. Commonly, these techniques are divided into two main groups: conventional and additive manufacturing techniques (6).

By the conventional techniques, some of the common procedures are solvent-casting/particulate-leaching, phase separation, freeze-drying, and gas infusion (12, 13). Solvent-casting/particulate-leaching comprises the addition of particles with specific diameter into a polymeric solution. Following, the solvent solution is allowed to evaporate, leaving the polymeric matrix with the salt particles embedded. This matrix is immersed in water and the salt particles are leached out, producing the scaffold. Phase separation technique is based on the production of a liquid–liquid separation induced by temperature decrease. One phase is a polymer-rich phase that solidifies and the other is a polymer-poor phase that crystallizes. The crystals formed on the polymer-poor phase are removed by sublimation, resulting in a highly porous structure. Additionally, bioactive molecules can be incorporated in that structure. Freeze-drying includes freezing a solution (polymer and glacial acetic or benzene) at low temperatures (−70°C to −80°C), causing the crystal formation. Then, the sample is freeze-dried: it is placed in a low-pressure chamber (primary drying) and the water is removed by desorption (second drying), leaving a 3D porous structure. However, these conventional techniques have a crucial drawback: the structure architecture is not controlled; thus, pore interconnectivity is not assured and the pore morphology (size, geometry, and distribution) is not uniform. Besides that, conventional techniques usually require human hand (less productive) and solvents or porogens to create pore structures, which are mostly toxic (residues may induce tissue responses) (14).

Additive manufacturing techniques have several advantages, allowing the production of scaffolds with precise geometries (computer-controlled fabrication), therefore, ensuring a high pore connectivity and a reliable constructs production (cf Fig. 1). Besides that, these techniques also permit using customized geometries, according to the patient specifications (14). Among others, additive manufacturing techniques include stereolithography (SLA), selective laser sintering (SLS), 3D printing (3DP), and fused deposition modelling (6). SLA comprises a low-power, high-focused UV laser that draws successive layers in a vat containing liquid photosensitive polymer. While the UV laser traces the polymer, it solidifies and, when that layer is completed, the platform is lowered, and the process is repeated until the structure is completed. SLS uses an infrared laser beam that selectively sinter the polymer and/or metal composite materials in a powder form, layer by layer, thereby obtaining a 3D structure. Initially, a powder material bed is deposited at high temperature to facilitate the sintering procedure, and the material not sintered underpins to the following layer. 3DP is based on the deposition of a liquid binder on powder material into successive cross sections, creating a 3D structure. This process starts with the powder deposition on the build chamber. Then, a print head deposits an adhesive liquid in selected regions of the powder bed, bonding the material and building a layer. After each layer, the platform is lowered and the process is repeated. The excess powder acts as a support to the next layer, and when the structure is finished, it is removed. Inkjet printing is very similar to 3DP, with one main difference; in 3DP, a liquid binder is deposited, whereas in inkjet printing melted material is deposited, mainly wax, resin, or thermoplastics. Besides, material deposition can be a continuous jet or drop-on-demand, that is, dropwise. Fused deposition modelling is based on the extrusion of (thermo)plastic or wax (usually supplied as filament or pellets) through a nozzle that draws the pretended built layer by layer. Resistive heaters covering the nozzle are used to maintain the material just above its melting point. This allows the material to easily flow through the nozzle, bonding to the layer below and achieving prompt solidification.

Fig. 1 Illustrative representation of the steps in additive manufacturing.

The example of temporomandibular joint disc

As above-mentioned, TE aims are to promote tissue repair and regeneration, or even replace it. Presently, almost every mammal tissue has been attempted to engineer: skin, liver, hearth, blood vessels, nerves, bone, and cartilage (4). Nevertheless, the cartilage regeneration keeps being a paramount challenge to be solved. One example is the TE of a fibrocartilaginous tissue, like synthetic temporomandibular disc, in order to restore the functionality of this joint.

Temporomandibular joint disorders (TMD) are the disturbances that most contribute to the orofacial chronic pain, usually coming from tissue degeneration or displacement of the TMJ disc. Symptoms include pain both in the joint and in the surrounding muscles, clicks, discomfort when moving the jaw (e.g., opening the mouth), and grit of teeth. Usually, these symptoms lead to pain over the complete face, neck, and head, and even in the ears. Common ear symptoms are otalgia, stuffiness, tinnitus, and hearing loss (15), with the first explanation of the association between TMD and aural symptoms being made more than 80 years ago (16). However, and due to the complexity of the structures, several hypotheses have been proposed to clarify that association (17). This led to the need for interdisciplinary treatment planning, gathering different health professionals (e.g., neurologists, ear-nose-and-throat physicians, and physical therapists) (18).

The TMJ is the articulation between the mandible and the temporal bone. The mandibular condyle fits into the glenoid fossa. A fibrocartilage disc is located between the mandible and the temporal bone, separating the joint in two cavities: superior and inferior (19). The joint is surrounded by a fibrous capsule which connects the edge of the TMJ disc, and that is reinforced by the lateral ligament and the accessory ligaments (sphenomandibular and stylomandibular ligaments). The TMJ allows the performing of three types of movement: 1) protraction and retraction – involves the anterior gliding motion of the mandibular condyle and articular disc relative to glenoid fossa; protraction movement combined with the hinge motion between the articular disk and the mandibular head allows the 2) elevation and depression of the mandible and 3) lateral and medial excursion of the mandible. TMJ disc is an essential component in the normal TMJ and has the following functions: 1) it distributes the intra-articular load; 2) it stabilizes the joints during translation; and 3) it decreases the wear of the articular surface (20, 21). It is a fibrocartilaginous tissue, with more collagen fibers than proteoglycans. Thus, it is a slightly compressible and very tough tissue, proper for areas where high pressures are applied. The majority of the TMD are successfully treated with reversible, conservative, and low-tech treatments such as education and counselling, therapeutic exercises, splint therapy, and pharmacotherapy (18, 22). However, when the TMJ disc is displaced, malformed, or damaged, it can induce serious internal pathologic processes and/or osteoarthritis (23, 24).

Regarding the TMJ disc, when it becomes morphologically damaged, surgeons may perform discectomy with or without autograft replacement (25). In fact, severe inflammation of TMJ presents a clinical challenge nowadays, as pharmacotherapy alone may not be effective and frequently surgical intervention is needed. The latter can range from autologous muscle grafts to TMJ prosthesis that also carries its own complications and failures, as exposed in Fig. 2, enabling TE as an optimal future treatment. In 1991, the first attempt to produce synthetic TMJ implants in Teflon (Proplast-Teflon®) caused progressive bone degeneration due to a foreign body response immunologically mediated, which caused severe consequences for the patients – graft versus host disease (26). By that time, a clear and complete understanding of TMJ disc characteristics, as well as its function, was lacking. Thus, TMJ disc characterization was extensively reviewed by Allen and Athanasiou (20). These authors reported that the TMJ disc can be divided into three distinct geometric regions: anterior band, posterior band, and intermediate zone. Additionally, it was stated that cells in the intermediate zone present a more chondrocytic nature and cells upon the periphery tend to become fibroblastic (27). This distribution is the result of a matrix structure dominated by collagen type I fibers generating a ring around the periphery with elastin fibers in the posterior band. These two characteristics are enough to understand the complexity of this tissue and the challenge that would be to use a TE or RM (TE&RM) approach to replicate it.

Fig. 2 A 19-year-old male patient with systemic arthritis before (panel a) and after (panel b) autologous temporal muscle was interposed between the joint that resulted into ankyloses within 6 months.

In the last years, some experiments were performed in TMJ disc TE&RM, with the goal of inducing tissue regeneration or complete disc replacement. A research group from the United States advanced with a study in which porcine ECM-based scaffolds were used to replace canine TMJ disc after discectomy and allow new functional tissue regeneration. This study showed promising results, suggesting that this type of scaffolds could be a valuable option for TMJ disc reconstruction, due to the similarity between native and regenerated tissue (28). In a study with rabbits, autologous bone marrow was added to a collagen sponge scaffold to induce tissue regeneration in perforated TMJ discs. Proceeding with an in situ TE approach positive results were obtained. Still, the motion exhibited by the rabbit model is evidently different to the one exhibited by the human joint (29).

Some preclinical studies have been performed in small animal models, but they fail in TMJ loading and anatomic differences. Others have been trying to identify appropriate suitable biomaterial, or biomaterial combination, for scaffold construction that could regenerate TMJ disc [e.g., PGA (polyglycolic acid), ePTFE (expanded polytetrafluoroethylene), or hydrogels] (30, 31). These experiments gave a significant contribution to the understanding of the TMJ disc replacement mechanism, but a convincing approach to the problem is still missing. Another experiment with interesting results used an elastomeric biodegradable polyester (poly glycerol-sebacate) as a porous scaffold material (25). As an interesting outcome, these authors showed that cell seeding density and culture time should be considered for in vitro trials.

Conclusion

In vivo trials for scaffold manufacturing that enhance the similarity to the native discs are still lacking. Once the TMJ disc is relatively avascular and does not naturally regenerate or repair itself in vivo, it has become a goal for TE. In fact, cartilage tissue has a very unique structure, stimulating several research groups to overcome this problem and achieve a proper regeneration. In oral and maxillofacial domains, a variety of techniques have been described in the literature, but no single technique has proven to be universally successful in reducing ankyloses recurrence and reproducing TMJ function. In fact, there is a lack of TE&RM approaches that lead to translational medicine.

Authors’ contributions

PM, DA, and LF wrote the manuscript. PM, CM, and NA discussed the context and contributed to the final manuscript.

Conflict of interest and funding

The authors have no conflict of interest to disclose.