Bioactive scaffolds integrated with micro-precise spatiotemporal delivery and in vivo degradation tracking for complex tissue regeneration

Three dimensional (3D) printing has emerged as an efficient tool for tissue engineering and regenerative medicine, given its advantages for constructing custom-designed scaffolds with tunable microstructure/physical properties. Here we developed a micro-precise spatiotemporal delivery system embedded in 3D printed scaffolds. PLGA microspheres (μS) were encapsulated with growth factors (GFs) and then embedded inside PCL microfibers that constitute custom-designed 3D scaffolds. Given the substantial difference in the melting points between PLGA and PCL and their low heat conductivity, μS were able to maintain its original structure while protecting GF’s bioactivities. Micro-precise spatial control of multiple GFs was achieved by interchanging dispensing cartridges during a single printing process. Spatially controlled delivery of GFs, with a prolonged release, guided formation of multi-tissue interfaces from bone marrow derived mesenchymal stem/progenitor cells (MSCs). To investigate efficacy of the micro-precise delivery system embedded in 3D printed scaffold, temporomandibular joint (TMJ) disc scaffolds were fabricated with micro-precise spatiotemporal delivery of CTGF and TGFβ3, mimicking native-like multiphase fibrocartilage. In vitro, TMJ disc scaffolds spatially embedded with CTGF/TGFβ3-μS resulted in formation of multiphase fibrocartilaginous tissues from MSCs. In vivo, TMJ disc perforation was performed in rabbits, followed by implantation of CTGF/TGFβ3-μS-embedded scaffolds. After 4 wks, CTGF/TGFβ3-μS embedded scaffolds significantly improved healing of the perforated TMJ disc as compared to the degenerated TMJ disc in the control group with scaffold embedded with empty μS. In addition, CTGF/TGFβ3-μS embedded scaffolds significantly prevented arthritic changes on TMJ condyles. In conclusion, our micro-precise spatiotemporal delivery system embedded in 3D printing may serve as an efficient tool to regenerate complex and inhomogeneous tissues.

Hypoxia increases expression of matrix metalloproteinase-3 in temporomandibular joint disc cells

The profiling and analysis of gene expression in rat temporomandibular joint disc tissue and its derived cells.

Analysis of the Collagen I and Fibronectin of Temporomandibular Joint Synovial Fluid and Discs

The Goat as a Model for Temporomandibular Joint Disc Replacement: Techniques for Scaffold Fixation

Purpose of this study was to investigate the expression of smooth muscle action-alpha (a-SMA) in native goat temporomandibular joint (TMJ) disc and in self-assembled construct of TMJ disc. Expression of a-SMA in native one-month-aged goat TMJ disc was examined by immunohistochemistry(IHC) method. TMJ disc cells were isolated from one-month-aged goat TMJ discs with 0.1% type I collagenase and passaged to P3 for tissue engineering. 5.5x10(6) cells were seeded into 5 mm diameter wells filled with 2% agarose gels in which 10µg/L IGF-I growth factor was added to stimulate mature process of self-assembled constructs of TMJ disc. Dimensions, forms and weights of constructs were measured respectively at the second, fourth, sixth and eighth week. IHC staining was used to examine the distribution of a-SMA in self-assembled constructs. The IHC staining for a-SMA was globally uniformly distributed in all four groups of self-assembled constructs and expressed the strongest in the second group. This experiment also confirmed that much more significant expression of a-SMA in self-assembled constructs than in the native TMJ disc tissue. Conclusively, a-SMA could take part in the regulating developmental process of engineered construct and may be used partially to explain the reason why TMJ disc self-assembled constructs become much smaller and denser during its growth and development due to its volume shrinkage.

The temporomandibular joint (TMJ) disc nutrient environment profoundly affects cell energy metabolism, proliferation, and biosynthesis. Due to technical challenges of in vivo measurements, the human TMJ disc extracellular nutrient environment under load, which depends on metabolic rates, solute diffusion, and disc morphometry, remains unknown. Therefore, the study objective was to predict the TMJ disc nutrient environment under loading conditions using combined experimental and computational modeling approaches. Specifically, glucose consumption and lactate production rates of porcine TMJ discs were measured under varying tissue culture conditions (n = 40 discs), and mechanical strain-dependent glucose and lactate diffusivities were measured using a custom diffusion chamber (n = 6 discs). TMJ anatomy and loading area were obtained from magnetic resonance imaging of healthy human volunteers (n = 11, male, 30 ± 9 y). Using experimentally determined nutrient metabolic rates, solute diffusivities, TMJ anatomy, and loading areas, subject-specific finite element (FE) models were developed to predict the 3-dimensional nutrient profiles in unloaded and loaded TMJ discs (unloaded, 0% strain, 20% strain). From the FE models, glucose, lactate, and oxygen concentration ranges for unloaded healthy human TMJ discs were 0.6 to 4.0 mM, 0.9 to 5.0 mM, and 0% to 6%, respectively, with steep gradients in the anterior and posterior bands. Sustained mechanical loading significantly reduced nutrient levels (P < 0.001), with a critical zone in which cells may die representing approximately 13.5% of the total disc volume. In conclusion, this study experimentally determined TMJ disc metabolic rates, solute diffusivities, and disc morphometry, and through subject-specific FE modeling, revealed critical interactions between mechanical loading and nutrient supply and metabolism for the in vivo human TMJ disc. The results suggest that TMJ disc homeostasis may be vulnerable to pathological loading (e.g., clenching, bruxism), which impedes nutrient supply. Given difficulties associated with direct in vivo measurements, this study provides a new approach to systematically investigate homeostatic and degenerative mechanisms associated with the TMJ disc.

Longitudinal study of temporomandibular joint disc status and craniofacial growth

The temporomandibular joint (TMJ) disc is a fibrocartilage tissue located between the mandibular condyle and the glenoid eminence, which is central to the TMJ functions. The TMJ disc is susceptible to irreparable degenerative changes or post-traumatic injuries, which can lead to the development of a disc-related disease. Scaffold-based tissue engineering offers the potential for regeneration and replacement of the damaged TMJ disc. The present review describes the biomaterials and manufacturing technologies used in scaffold-based TMJ disc engineering strategies and comprehensively evaluates the advantages and disadvantages of each strategy. As an understanding of the extracellular matrix (ECM) is fundamental for succesful TMJ disc tissue engineering, this review defines the key properties and roles of the TMJ disc ECM. Compared with the natural disc, the mechanical properties of the tissue-engineered TMJ disc are not satisfactory. Additionally, the in vivo durability of engineered discs and their long-term impact on the entire TMJ remain to be studied, especially in large-animal preclinical trials.

The aquaporins (AQPs) are a family of hydrophobic membrane channel proteins that are expressed in many epithelial, endothelial and other tissues and that participate in many physiological and pathological processes. In particular aquaporin-1 (AQP1), a member of this family proteins, is expressed by the masseter and infrahyoid muscles, meckel’s cartilage and submandibular gland. Literature data on the role and the localization of AQP1 in articular joints are very poor, and none providing data on the normal temporomandibular joint (TMJ) disc. Accordantly, the present paper prompted this immunohistochemical and western blotting investigation, on the presence and distribution of AQP1 in human TMJ discs without any degenerative changes, in order to elucidate further aspects of the TMJ homeostasis system and disc tribology. Twelve TMJ discs, were obtained bilaterally from six cadavers and they were processed for immunohistochemical and Western Blot analysis. The results showed an AQP1 immunoexpression in few fibroblasts-like cells and fibrochondrocytes of normal human TMJ discs demonstrating a constitutive expression of this protein. No difference between the disc regions was seen in normal specimens. Western blot analysis of disc samples confirm the physiological AQP1 expression. In this view it appears reasonable a physiological expression of AQP1 by TMJ fibrocartilage cells and interpretable as a physiological mechanism of tissue homeostasis. Further future interesting studies could be conducted to elucidate the full AQPs profile in TMJ pathological disc investigating their change of the expression patterns during TMJ disc diseases.

Defects and dysfunctions of temporomandibular joint (TMJ) disc are responsible for the majority of TMJ diseases. Current treatments in this matter are usually short-term and only palliative, thus an alternative treatment that offers long-lasting repair is in great demand. In recent years great attempts have been made to prepare an ideal scaffold, which best resembles the native TMJ disc in characteristics such as mechanical, physical and biological properties. This narrative review focuses on developments of the recent ten years in fabrication of scaffolds using decellularized tissues, natural and synthetic biomaterials for regeneration of TMJ disc and compared their properties. PubMed and Google Scholar databases were searched using the following keywords ("TMJ" OR "temporomandibular joint" OR "TMD" OR "temporomandibular disease") AND ("scaffold" OR "hydrogels"). Randomized controlled trials, randomized clinical trials, case-controls, case reports, and animal studies were included. Comments, systematic reviews, meta-analyses, and non-English papers were excluded. The study concluded that hybrid scaffolds have exhibited favorable cell attachment and proliferation. Synthetic scaffolds have shown promise in providing better control over structural properties; however, additional processes are often required to provide biomimetic cell signaling. While there is still much to learn about the ideal scaffold for TMJ disc regeneration, both natural and synthetic scaffolds have shown promise in achieving the functional, structural, biological, and mechanical properties of a native TMJ disc.

The apoptosis is the programmed cell death, a distinct process compared to cellular necrosis, which plays an important role in both human embryonic development and adult tissue homeostasis. Apoptosis represents a physiological form of cell death. The role of apoptosis is to remove harmful, damaged or unwanted cells without inducing inflammatory response by the release of cell contents [1]. Apoptosis is activated through two principal signaling pathways: intrinsic and extrinsic, both of which are potential anticancer therapeutic targets. In contrast to necrosis, which is a form of cell death resulting from an acute cellular stress or trauma, apoptosis is a death orderly and regular, does not induce inflammation, requires energy (ATP) and generally lead to an advantage during the body life. Besides its importance as a biological phenomenon, it has acquired a huge medical value, since imperfect process of apoptosis covers many diseases. Excessive activity can cause trouble by apoptotic cell loss (see, for example neurodegenerative diseases such as Parkinson’s disease), while a weak apoptosis may involve uncontrolled cell growth in malignancy mechanism [2]. Apoptosis is the most common mechanism by which the body eliminates damaged or unneeded cells, without local inflammation from leakage of cell contents. Cells that are undergoing apoptosis exhibit a characteristic pattern of morphologic changes, including cell shrinkage, condensation, fragmentation of the nucleus and bubbling of the plasma membrane, known as “blebbing,” and chromatin condensation and nucleosomal fragmentation [1,3]. Apoptosis functions through two main, alternative pathways: death receptor-mediated (or extrinsic) and mitochondria-dependent (or intrinsic). The former pathway is initiated by ligation of specific death receptors by their ligands. The main death receptors—Fas and tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptors R1 and R2—induce cell death following ligation with Fas ligand (FasL) or TRAIL, respectively [4-7]. Ligation of TRAIL R1 by TNFα also induces apoptosis after inhibition of nuclear factor kappa-B (NF-kB). Fas ligation by FasL is followed by recruitment of FADD (Fas-associated via death domain) and subsequently of caspase 8. This process gives rise to caspase 8 activation, which can be inhibited by the anti-apoptotic molecule FLICE inhibitory protein (Flip). Caspase 8 induces apoptosis by directly activating caspase 3 or by cleaving bid (BH3 interacting domain death agonist), resulting in mitochondrial dysfunction and subsequent release of cytochrome c and activation of caspases 9 and 3. Caspase 3 promotes the typical apoptosis features, including DNA fragmentation and cell death in several tissues [8,9]. It is unclear whether repetitive loading and apoptosis are quantitatively related in any type of cartilage. Several studies have demonstrated the crucial function of apoptotic mechanisms in intervertebral disc degeneration, and the involvement of apoptosis in various conditions associated with intervertebral disc degeneration has been thoroughly explored [10]. However, apoptosis has been documented to have a central role not only in intervertebral disc degeneration [10]. Although a number of features of temporomandibular joint (TMJ) disc degeneration have been thoroughly studied, data on the involvement of apoptotic mechanisms and their mediators are few and quite recent; indeed most of the research conducted on disc apoptosis has focused on the intervertebral disc. TMJ disc degeneration is believed to be a consequence of mechanical and biological events affecting the equilibrium between matrix synthesis and degradation. According to this hypothesis loss of cellularity, collagen fibre fragmentation and TMJ disc tears and clefts would all depend on excessive apoptosis, a situation where collagen fibre degradation is not offset by synthesis of new fibres because of apoptosis-induced cell loss. The findings correlating TMJ disc internal derangement and apoptosis largely come from animal models [11]. However, our group has extensively analyzed programmed cell death in human TMJ discs with and without internal derangement [12,13]. We have advanced Corresponding author.

The temporomandibular joint (TMJ) disc is a heterogeneous fibrocartilaginous tissue positioned between the mandibular condyle and glenoid fossa of the temporal bone, with important roles in TMJ functions. Tissue engineering TMJ discs has emerged as an alternative approach to overcoming limitations of current treatments for TMJ disorders. However, the anisotropic collagen orientation and inhomogeneous fibrocartilaginous matrix distribution present challenges in the tissue engineering of functional TMJ discs. Here, we developed 3-dimensional (3D)–printed anatomically correct scaffolds with region-variant microstrand alignment, mimicking anisotropic collagen alignment in the TMJ disc and corresponding mechanical properties. Connective tissue growth factor (CTGF) and transforming growth factor beta 3 (TGFβ3) were then delivered in the scaffolds by spatially embedding CTGF- or TGFβ3-encapsulated microspheres (µS) to reconstruct the regionally variant fibrocartilaginous matrix in the native TMJ disc. When cultured with human mesenchymal stem/progenitor cells (MSCs) for 6 wk, 3D-printed scaffolds with CTGF/TGFβ3-µS resulted in a heterogeneous fibrocartilaginous matrix with overall distribution of collagen-rich fibrous structure in the anterior/posterior (AP) bands and fibrocartilaginous matrix in the intermediate zone, reminiscent of the native TMJ disc. High dose of CTGF/TGFβ3-µS (100 mg µS/g of scaffold) showed significantly more collagen II and aggrecan in the intermediate zone than a low dose (50 mg µS/g of scaffold). Similarly, a high dose of CTGF/TGFβ3-µS yielded significantly higher collagen I expression in the AP bands compared with the low-dose and empty µS. From stress relaxation tests, the ratio of relaxation modulus to instantaneous modulus was significantly smaller with CTGF/TGFβ3-µS than empty µS. Similarly, a significantly higher coefficient of viscosity was achieved with the high dose of CTGF/TGFβ3-µS compared with the low-dose and empty µS, suggesting the dose effect of CTGF and TGFβ3 on fibrocartilage formation. Together, our findings may represent an efficient approach to engineering the TMJ disc graft with anisotropic scaffold microstructure, heterogeneous fibrocartilaginous matrix, and region-dependent viscoelastic properties.

Objective: To summarize the clinical manifestation and treatment of temporomandibular joint (TMJ) disc ossification, providing reference for clinical diagnosis and treatment of TMJ disc ossification. Methods: From January 2006 to January 2018, 4 patients with TMJ disc ossification (2 males and 2 females, aged 20-55 years with an average age of 35.5 years) which were admitted to the Department of Oral and Maxillofacial Surgery, Shenzhen Second People's Hospital were analyzed retrospectively. Ossification of TMJ disc was found in 4 cases during TMJ surgery. Two cases underwent partial ossification resection plus disc reduction and anchorage, and two cases underwent discectomy plus temporalis myofascial flap replacement. The causes, clinical manifestations and surgical effects of TMJ disc ossification were analyzed by comparing the maximal interincisal opening, visual analogue scale (VAS) score and MRI imaging indexes before and after operation. Results: The history of anterior disc displacement of TMJ in 4 patients was long (average 11.5 years). In clinic, TMJ disc ossification was characterized by TMJ pain and limitation of mouth opening. The maximal interincisal opening was (32.1±6.1) mm and the VAS score was (7.3±0.4) before operation. MRI showed that the displaced discs of the affected sides were displaced and the condyle bones were worn. During the operation, ossification of TMJ discs was found yellow and hard, and the original elasticity was lost. Pathologic findings showed that the TMJ disc cartilage were ossified to osteoid tissue. Under the microscope, bone cells scattered around the bone cells and red trabecular bone were seen, and there were bone trabecula formed. In a follow-up of one year, TMJ pain was significantly decreased [VAS: (1.7±0.2)], and the maximal interincisal opening was (38.5±2.2) mm. MRI showed that the TMJ disc returned to normal position, and the sign of repairing and reconstruction of condyle bone could be found. Conclusions: Long term displacement of TMJ disc may cause ossification with pain and limitation of interincisal opening. According to the degree and extent of ossification, partial ossification plus disc reduction and anchorage or discectomy plus temporalis myofascial flap replacement is feasible, and the clinical effects are satisfactory.

Residents' journal review