Anisotropic Solute Diffusion Tensor in Porcine TMJ Discs Measured by FRAP with Spatial Fourier Analysis

A new method solely based on spatial Fourier analysis (SFA) was developed to completely determine a two-dimensional (2D) anisotropic diffusion tensor in fibrous tissues using fluorescence recovery after photobleaching (FRAP). The accuracy and robustness of this method was validated using computer-simulated FRAP experiments. This method was applied to determine the region-dependent anisotropic diffusion tensor in porcine temporomandibular joint (TMJ) discs. The average characteristic diffusivity of 4 kDa FITC-Dextran across the disc was 26.05 ± 4.32 μm²/s which is about 16% of its diffusivity in water. In the anteroposterior direction, the anterior region (30.99 ± 5.93 μm²/s) had significantly higher characteristic diffusivity than the intermediate region (20.49 ± 5.38 μm²/s) and posterior region (20.97 ± 2.46 μm²/s). The ratio of the two principal diffusivities represents the anisotropy of the diffusion and ranged between 0.45 and 0.51 (1.0 = isotropic). Our results indicated that the solute diffusion in TMJ discs is inhomogenous and anisotropic. These findings suggested that diffusive transport in the TMJ disc is dependent on tissue composition (e.g., water content) and structure (e.g., collagen orientation). This study provides a new method to quantitatively investigate the relationship between solute transport properties and tissue composition and structure.     

Design Characteristics for the Tissue Engineering of Cartilaginous Tissues

Tissues like the temporomandibular joint (TMJ) disc and the knee meniscus are often mistakenly viewed as a tantamount to hyaline cartilage, largely due to the absence of a comprehensive understanding of the distinguishing properties of cartilaginous tissues. Because of this confusion, fibrocartilaginous tissue engineering attempts may not be based on suitable experimental designs. Fibrocartilaginous tissues are markedly different than hyaline cartilage; however, the dearth of knowledge related to their cellular and biochemical composition, as well as their biomechanical characteristics, is stunning. Hyaline articular cartilage is exclusively composed of chondrocytes that produce primarily type II collagen, whereas the TMJ disc and the knee meniscus have a mixed cell population of fibroblasts and cells similar to chondrocytes, which predominantly secrete type I collagen. Additionally, fibrocartilaginous tissues have a low glycosaminoglycan content, a low compressive modulus, and a high tensile modulus when compared to hyaline cartilage. Therefore, it is crucial for fibrocartilaginous tissue engineering attempts to be tissue-specific, utilizing the knowledge of the distinct and unique properties of these tissues. At the same time, advances and insights related to the science and engineering aspect of hyaline cartilage regeneration must be carefully considered for the in vitro engineering of fibrocartilaginous tissues.     

Combined use of chondroitinase-ABC,TGF-β1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility.     

Tissue Engineering of the TMJ disc: a review

The potential impact of a tissue-engineered temporomandibular joint (TMJ) disc is immense. Currently, patients suffering from a severely dysfunctional TMJ have few options. Facing the general lack of safe, effective TMJ disc implants, many patients undergo discectomy, a procedure that removes the injured TMJ disc in hopes of reducing debilitating symptoms associated with severe TMJ disorders. This procedure may not be ideal as the TMJ is left without an important functional component. Tissue engineering is a promising approach for the creation of viable, effective implants. The first attempt to investigate TMJ disc cells on a biomaterial was conducted in 1991. The first TMJ tissue-engineered constructs to be tested biochemically and biomechanically were formed in 1994; however, in examining this study in retrospect, it is clear how little TMJ knowledge was available at that time. Within the last 10 to 15 years, multiple studies have investigated critical TMJ disc characteristics, and while this characterization is not complete, these data have created a solid foundation for tissue-engineering research. Thus, the last 5 years have yielded core studies investigating the principal elements of tissue engineering: scaffold, cell source, and biological/biomechanical stimuli. Although TMJ disc tissue engineering is still in its formative years, its future is quite promising. Key studies are now being conducted that will assist in the establishment of a solid TMJ disc tissue-engineering approach. As the challenges of tissue engineering are faced and met, the ultimate goal of creating a functional biological implant nears.     

Maturational growth of self-assembled, functional menisci as a result of TGF-β1 and enzymatic chondroitinase-ABC stimulation

Replacement of the knee meniscus requires a material possessing adequate geometrical and biomechanical properties. Meniscal tissue engineering attempts have been unable to produce tissue with collagen content and biomechanical properties, particularly tensile properties, mimicking native menisci. In an effort to obtain the geometric properties and the maturational growth necessary for the recapitulation of biochemical and, thus, biomechanical properties, a scaffoldless cell-based system, the self-assembly process, was used in conjunction with the catabolic enzyme chondroitinase-ABC and TGF-β1. We show that combinations of these agents resulted in maturational growth as evidenced by synergistic enhancement of the radial tensile modulus by 5-fold and the compressive relaxation modulus by 68%, and additive increases of the compressive instantaneous modulus by 136% and Col/WW by 196%. This study shows that tissue engineering can produce a biomaterial that is on par with the biochemical and biomechanical properties of native menisci.     

工程化颞下颌关节盘组织的细胞源问题:现状与展望

目的是为学者探索适当的颞下颌关节盘组织工程种子细胞提供参考。目前颞下颌关节盘组织工程正处于起步阶段,而细胞来源是制约关节盘组织工程发展的主要因素之一,本文概述了关节盘细胞、软骨细胞、皮肤成纤维细胞、骨髓基质干细胞、脂肪干细胞和胚胎干细胞等6种细胞作为关节盘组织工程细胞源的可行性,以便寻找一种合适的细胞源。     

Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model

The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering method.     

Evaluating Anisotropic Properties in the Porcine Temporomandibular Joint Disc Using Nanoindentation

The objective of this study was to determine the viscoelastic properties present within the intermediate zone of the porcine temporomandibular joint (TMJ) disc using nanoindentation. A 50-μm conospherical indenter tip using a displacement-controlled ramp function with a 600 nm/s loading and unloading rate, a 3000-nm peak displacement with a holding period of 30 s was used to indent the samples. Experimental load-relaxation tests were performed on the TMJ disc to determine the response in three different directions; the mediolateral, anteroposterior, and articular surface directions. The experimental data were analyzed using a generalized Maxwell model to obtain values for short- and long-time relaxation modulus and of material time constants. The short time relaxation modulus E _I values were 180.92, 64.99, and 487.77 kPa for testing done on the articular surface, mediolateral, and anteroposterior directions, respectively. Corresponding values for the long-time relaxation modulus E _∞ were 45.9, 14.97, and 133.5 kPa. The method confirmed anisotropy present within the central intermediate zone of the porcine TMJ disc due to the directional orientation of the collagen fibers.     

Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model

The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering method.     

A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc

Tissue-engineered temporomandibular joint (TMJ) discs offer a viable treatment option for patients with severe joint internal derangement. To date, only a handful of TMJ tissue engineering studies have been carried out and all have incorporated the use of synthetic scaffold materials. These current scaffolds have shown limited success in recapitulating morphological and functional aspects of the native disc tissue. The present study is the first to investigate the potential of a xenogeneic scaffold for use in tissue engineering the TMJ disc. The effects of decellularization agents on the disc's mechanical properties were assessed using three common decellularization protocols: Triton X-100, sodium dodecyl sulfate (SDS) and an acetone/ethanol solution. Decellularized scaffolds were subsequently characterized through cyclic mechanical testing at physiologically relevant frequencies to determine which chemical agent most accurately preserved the native tissue properties. Results have shown that porcine discs treated with SDS most closely matched the energy dissipation capabilities and resistance to deformation of the native tissue. Treatments using Triton X-100 caused the resultant tissue to become relatively softer with inferior energy dissipation capabilities, while treatment using acetone/ethanol led to a significantly stiffer and dehydrated material. These findings support the potential of a porcine-derived scaffold decellularized by SDS as a xenograft for TMJ disc reconstruction.     

Effects of Initial Cell Seeding Density for the Tissue Engineering of the Temporomandibular Joint Disc

Tissue engineering may provide a better treatment modality for postoperative discectomy patients. The TMJ disc is an ideal candidate for tissue engineering approaches because of its lack of an intrinsic regenerative ability. Unfortunately, basic knowledge related to TMJ disc tissue engineering is still at an infancy level and not on par to that related to articular cartilage tissue engineering. The objective of this study was to examine the effects of initial cell density of TMJ disc cells seeded in nonwoven poly-glycolic acid (PGA) scaffolds on the biochemical and biomechanical properties of constructs examined at 0, 3, and 6 weeks after seeding. Low, medium, and high seeding densities were chosen to be 15, 30, and 120 million cells per ml of scaffold, which were seeded using a spinner flask. Significant differences were found temporally and as a function of seeding density in morphology, total collagen, GAG content, and permeability of the constructs, but not in aggregate modulus. The high seeding density group outperformed the low and medium groups in collagen and GAG content at all time points measured. The high-density group produced a total of 55.37 ± 3.56 μg of collagen per construct, maintained 15.77 ± 1.86 μg of GAG per construct, and only shrunk to 50% of the original scaffold size. Permeability of the constructs at 6 weeks was decreased by 70% compared to 0 weeks.     

Immunolocalization and expression of lubricin in the bilaminar zone of the human temporomandibular joint disc

Lubricin, which is a boundary joint lubricant, was investigated immunohistochemically in the bilaminar zone (BZ) of the human temporomandibular joint (TMJ), without any degenerative changes. Immunohistochemistry for lubricin detection was carried out on 33 TMJ discs obtained from 17 cadavers. Sections were incubated with diluted rabbit polyclonal anti-lubricin antibody and scored according to the percentage of lubricin immunopositive cells. Three different TMJ disc tissue compartments were analyzed, namely: the upper lamina, the inferior lamina and the loose connective tissue in the space between the laminae. The Mann-Whitney U test was used to compare protein expression (lubricin) among disc specimens’ regions. Staining was noted within the TMJ disc cell populations in every disc tissue sample, with almost every cell immunolabeled by the lubricin antibody. The number of disc cells immunolabeled was almost the same in the 3 bilaminar zone regions. Positive extracellular matrix staining was also seen. The results of the present study suggest that lubricin is expressed in the TMJ disc bilaminar zone. Lubricin may have a role in normal disc posterior attachment physiology through the prevention of cellular adhesion as well as providing lubrication during normal bilaminar zone function.     

Lubrication of the Temporomandibular Joint

Although tissue engineering of the temporomandibular joint (TMJ) structures is in its infancy, tissue engineering provides the revolutionary possibility for treatment of temporomandibular disorders (TMDs). Recently, several reviews have provided a summary of knowledge of TMJ structure and function at the biochemical, cellular, or mechanical level for tissue engineering of mandibular cartilage, bone and the TMJ disc. As the TMJ enables large relative movements, joint lubrication can be considered of great importance for an understanding of the dynamics of the TMJ. The tribological characteristics of the TMJ are essential for reconstruction and tissue engineering of the joint. The purpose of this review is to provide a summary of advances relevant to the tribological characteristics of the TMJ and to serve as a reference for future research in this field. This review consists of four parts. Part 1 is a brief review of the anatomy and function of the TMJ articular components. In Part 2, the biomechanical and biochemical factors associated with joint lubrication are described: the articular surface topology with microscopic surface roughness and the biomechanical loading during jaw movements. Part 3 includes lubrication theories and possible mechanisms for breakdown of joint lubrication. Finally, in Part 4, the requirement and possibility of tissue engineering for treatment of TMDs with degenerative changes as a future treatment regimen will be discussed in a tribological context.     

Biomechanical Properties of Transplanted Xenogenic Meniscal Tissue

The clinical repair approaches to meniscal lesions include total or subtotal meniscectomy, transplantation, and tissue engineering. The investigations found that the transplanted xenogenic meniscal tissues, which were treated by60 Co irradiation and deep freezing, maybe one of the effective approaches. In this paper, we evaluated the biomechanical properties of the transplanted xenogenic meniscal tissues at postoperative 1 year. In vitro tensile and compressive tests are performed to compare the properties of tensile elasticity, tensile strength, and compressive elasticcity,between three groups: RAB group of normal rabbit meniscus tissue, Allo group of transplanted allograft meniscal tissue, and Xeno group of transplanted xenogenic meniscal tissue. The meniscus of the Xeno group showed similar tension and compression modulus as the native rabbit meniscus without significant difference(p0.05),and the tension strength of both the Xeno group and the Allo group were less than that of the native rabbit meniscus(0.05p0.1). No significant difference was found between the Xeno group and the Allo group with regard to each biomechanical parameter(p0.05). These base studies will be helpful in future transplantation and tissue engineering efforts.     

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-β1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790 kPa), relaxed compressive moduli (120 kPa) and Young's moduli (1.87 MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-β1 treatment increased collagen deposition ～20%, compressive stiffness ～130% and Young's modulus ～170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-β1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.     

Stem cell-based meniscus tissue engineering

Knee meniscus, a fibrocartilaginous tissue, is characterized by heterogeneity in extracellular matrix (ECM) and biomechanical properties, and critical for orthopedic stability, load transmission, shock absorption, and stress distribution within the knee joint. Most damage to the meniscus cannot be effectively healed by the body due to its partial avascular nature; thus, damage caused by injury or age impairs normal knee function, predisposing patients to osteoarthritis. Meniscus tissue engineering offers a possible solution to this problem by generating replacement tissue that may be implanted into the defect site to mimic the function of natural meniscal tissue. To address this need, a multiporous, multilamellar meniscus was formed using silk protein scaffolds and stem cells. The silk scaffolds were seeded with human bone marrow stem cells and differentiated over time in chondrogenic culture in the presence of transforming growth factor-beta 3 to generate meniscus-like tissue in vitro. High cellularity along with abundant ECM leading to enhanced biomechanics similar to native tissue was found. Higher levels of collagen type I and II, sulfated glycosaminoglycans along with enhanced collagen 1-α1, aggrecan, and SOX9 gene expression further confirmed differentiation and matured cell phenotype. The results of this study are a step forward toward biomechanically competent meniscus engineering, reconstituting both form and function of the native meniscus.     

Serum neutralization of SARS coronavirus 2 Omicron sublineages BA.1 and BA.2 and cellular immune responses 3 months after booster vaccination

ObjectivesWe investigated serum neutralizing activity against BA.1 and BA.2 Omicron sublineages and T cell response before and 3 months after administration of the booster vaccine in healthcare workers (HCWs).MethodsHCWs aged 18–65 years who were vaccinated and received booster doses of the BNT162b2 vaccine were included. Anti–SARS coronavirus 2 IgG levels and cellular response (through interferon γ ELISpot assay) were evaluated in all participants, and neutralizing antibodies against Delta, BA.1, and BA.2 were evaluated in participants with at least one follow-up visit 1 or 3 months after the administration of the booster dose.ResultsAmong 118 HCWs who received the booster dose, 102 and 84 participants attended the 1-month and 3-month visits, respectively. Before the booster vaccine dose, a low serum neutralizing activity against Delta, BA.1, and BA.2 was detectable in only 39/102 (38.2%), 8/102 (7.8%), and 12/102 (11.8%) participants, respectively. At 3 months, neutralizing antibodies against Delta, BA.1, and BA.2 were detected in 84/84 (100%), 79/84 (94%), and 77/84 (92%) participants, respectively. Geometric mean titres of neutralizing antibodies against BA.1 and BA.2 were 2.2-fold and 2.8-fold reduced compared with those for Delta. From 1 to 3 months after the administration of the booster dose, participants with a recent history of SARS coronavirus 2 infection (n = 21/84) had persistent levels of S1 reactive specific T cells and neutralizing antibodies against Delta and BA.2 and 2.2-fold increase in neutralizing antibodies against BA.1 (p 0.014). Conversely, neutralizing antibody titres against Delta (2.5-fold decrease, p < 0.0001), BA.1 (1.5-fold, p 0.02), and BA.2 (2-fold, p < 0.0001) declined from 1 to 3 months after the administration of the booster dose in individuals without any recent infection.DiscussionThe booster vaccine dose provided significant and similar response against BA.1 and BA.2 Omicron sublineages; however, the immune response declined in the absence of recent infection.     

Biomaterial-mediated delivery of degradative enzymes to improve meniscus integration and repair

Endogenous repair of fibrous connective tissues is limited, and there exist few successful strategies to improve healing after injury. As such, new methods that advance repair by promoting cell growth, extracellular matrix (ECM) production, and tissue integration would represent a marked clinical advance. Using the meniscus as a test platform, we sought to develop an enzyme-releasing scaffold that enhances integrative repair. We hypothesized that the high ECM density and low cellularity of native tissue present physical and biological barriers to endogenous healing, and that localized collagenase treatment might expedite cell migration to the wound edge and tissue remodeling. To test this hypothesis, we fabricated a delivery system in which collagenase was stored inside electrospun poly(ethylene oxide) (PEO) nanofibers and released upon hydration. In vitro results showed that partial digestion of the wound interface improved repair by creating a microenvironment that facilitated cell migration, proliferation and matrix deposition. Specifically, treatment with high-dose collagenase led to a 2-fold increase in cell density at the wound margin and a 2-fold increase in integrative tissue compared to untreated controls at 4 weeks (P ? 0.05). Furthermore, when composite scaffolds containing both collagenase-releasing and structural fiber fractions were placed inside meniscal tears in vitro, enzyme release acted locally and resulted in a positive cellular response similar to that of global treatment with aqueous collagenase. This innovative approach to targeted enzyme delivery may aid the many patients that exhibit meniscal tears by promoting integration of the defect, thereby circumventing the pathologic consequences of partial meniscus removal, and may find widespread application in the treatment of injuries to a variety of dense connective tissues.