Passage and reversal effects on gene expression of bovine meniscal fibrochondrocytes

The knee meniscus contains a mixed population of cells that exhibit fibroblastic as well as chondrocytic characteristics. Tissue engineering studies and future therapies for the meniscus require a large population of cells that are seeded on scaffolds. To achieve this, monolayer expansion is often used as a technique to increase cell number. However, the phenotype of these cells may be significantly different from that of the primary population. The objective of this study was to investigate changes in meniscal fibrochondrocytes at the gene expression level over four passages using quantitative real-time reverse transcriptase polymerase chain reaction. Cells from the inner two-thirds of bovine medial menisci were used. Four extracellular matrix (ECM) molecules, commonly found in the meniscus, were investigated, namely collagen I, collagen II, aggrecan and cartilage oligomeric matrix protein (COMP). In addition, primary and passaged meniscus fibrochondrocytes were placed on surfaces coated with collagen I or aggrecan protein to investigate whether any gene expression changes resulting from passage could be reversed. Collagen I expression was found to increase with the number of passages, whereas collagen II and COMP expression decreased. Collagen I and aggrecan surface coatings were shown to downregulate and upregulate collagen I and COMP expression levels, respectively, in passaged cells. However, decreases in collagen II expression could not be reversed by either protein coating. These results indicate that although monolayer expansion results in significant changes in gene expression in meniscal fibrochondrocytes, protein coatings may be used to regain the primary cell expression of several ECM molecules

Tensile Properties, Collagen Content, and Crosslinks in Connective Tissues of the Immature Knee Joint

The major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by biochemical composition. A thorough understanding of the structure-function relationships of the connective tissues of the knee joint is needed to provide design parameters for efforts in tissue engineering.The objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of condylar cartilage, patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to anterior and posterior cruciate ligaments in humans, respectively), medial and lateral collateral ligaments, and patellar ligament from immature bovine calves. Tensile stiffness and strength were greatest in the menisci and patellar ligament, and lowest in the hyaline cartilages and cruciate ligaments; these tensile results reflected trends in collagen content. Pyridinoline crosslinks were found in every tissue despite the relative immaturity of the joints, and significant differences were observed among tissues. Notably, for the cruciate ligaments and patellar ligament, crosslink density appeared more important in determining tensile stiffness than collagen content.To our knowledge, this study is the first to examine tensile properties, collagen content, and pyridinoline crosslink abundance in a direct head-to-head comparison among all of the major connective tissues of the knee. This is also the first study to report results for pyridinoline crosslink density that suggest its preferential role over collagen in determining tensile stiffness for certain tissues

Engineering functional anisotropy in fibrocartilage neotissues

The knee meniscus, intervertebral disc, and temporomandibular joint (TMJ) disc all possess complex geometric shapes and anisotropic matrix organization. While these characteristics are imperative for proper tissue function, they are seldom recapitulated following injury or disease. Thus, this study's objective was to engineer fibrocartilages that capture both gross and molecular structural features of native tissues. Self-assembled TMJ discs were selected as the model system, as the disc exhibits a unique biconcave shape and functional anisotropy. To drive anisotropy, 50:50 co-cultures of meniscus cells and articular chondrocytes were grown in biconcave, TMJ-shaped molds and treated with two exogenous stimuli: biomechanical (BM) stimulation via passive axial compression and bioactive agent (BA) stimulation via chondroitinase-ABC and transforming growth factor-β1. BM + BA synergistically increased Col/WW, Young's modulus, and ultimate tensile strength 5.8-fold, 14.7-fold, and 13.8-fold that of controls, respectively; it also promoted collagen fibril alignment akin to native tissue. Finite element analysis found BM stimulation to create direction-dependent strains within the neotissue, suggesting shape plays an essential role toward driving in vitro anisotropic neotissue development. Methods used in this study offer insight on the ability to achieve physiologic anisotropy in biomaterials through the strategic application of spatial, biomechanical, and biochemical cues

Thyroid hormones enhance the biomechanical functionality of scaffold-free neocartilage

IntroductionThe aim of this study was to investigate the effects of thyroid hormones tri-iodothyronine (T3), thyroxine (T4), and parathyroid hormone (PTH) from the parathyroid glands, known to regulate the developing limb and growth plate, on articular cartilage tissue regeneration using a scaffold-free in vitro model.MethodsIn Phase 1, T3, T4, or PTH was applied during weeks 1 or 3 of a 4-week neocartilage culture. Phase 2 employed T3 during week 1, followed by PTH during week 2, 3, or weeks 2 to 4, to further enhance tissue properties. Resultant neotissues were evaluated biochemically, mechanically, and histologically.ResultsIn Phase 1, T3 and T4 treatment during week 1 resulted in significantly enhanced collagen production; 1.4- and 1.3-times untreated neocartilage. Compressive and tensile properties were also significantly increased, as compared to untreated and PTH groups. PTH treatment did not result in notable tissue changes. As T3 induces hypertrophy, in Phase 2, PTH (known to suppress hypertrophy) was applied sequentially after T3. Excitingly, sequential treatment with T3 and PTH reduced expression of hypertrophic marker collagen X, while yielding neocartilage with significantly enhanced functional properties. Specifically, in comparison to no hormone application, these hormones increased compressive and tensile moduli 4.0-fold and 3.1-fold, respectively.ConclusionsThis study demonstrated that T3, together with PTH, when applied in a scaffold-free model of cartilage formation, significantly enhanced functional properties. The novel use of these thyroid hormones generates mechanically robust neocartilage via the use of a scaffold-free tissue engineering model

Characterization of the Temporomandibular Joint Disc Complex in the Yucatan Minipig

The temporomandibular joint (TMJ) disc complex (i.e., the TMJ disc and its six attachments) is crucial to everyday functions such as mastication and speaking. The TMJ can be afflicted by many conditions, including disc displacement and defects. Pathologies of the TMJ disc complex most commonly present first as anterior disc displacement, which the field hypothesizes may implicate the two posterior attachments. As a result of anterior disc displacement, defects may develop in the lateral disc complex. Tissue engineering is poised to improve treatment paradigms for these indications of the TMJ disc complex by engineering biomimetic implants, but, first, gold-standard design criteria for such implants should be established through characterization studies. This study's objective was to characterize the structural, mechanical, biochemical, and crosslinking differences among the two posterior attachments and the lateral disc in the Yucatan minipig, a well-accepted TMJ animal model. In tension, it was found that the posterior inferior attachment (PIA) was significantly stiffer and stronger by 2.13 and 2.30 times, respectively, than the posterior superior attachment (PSA). It was found that collagen in both attachments was primarily aligned mediolaterally; however, the lateral disc was much more aligned and anisotropic than either attachment. Among the three locations, the PSA exhibited the greatest degree of heterogeneity and highest proportion of fat vacuoles. The PIA and lateral disc were 1.93 and 1.91 times more collagenous, respectively, by dry weight (DW) than the PSA. The PIA also exhibited 1.78 times higher crosslinking per DW than the PSA. Glycosaminoglycan per DW was significantly higher in the lateral disc by 1.48 and 5.39 times than the PIA and PSA, respectively. Together, these results establish design criteria for tissue-engineering of the TMJ disc complex and indicate that the attachments are less fibrocartilaginous than the disc, while still significantly contributing to the mechanical stability of the TMJ disc complex during articulation. These results also support the biomechanical function of the PIA and PSA, suggesting that the stiffer PIA anchors the disc to the mandibular condyle during articulation, while the softer PSA serves to allow translation over the articular eminence. Impact Statement Characterization of the temporomandibular joint (TMJ) disc complex (i.e., the disc and its attachments) has important implications for those aiming to tissue-engineer functional replacements and can help elucidate its biomechanical function. For example, the findings shown here suggest that the stiffer posterior inferior attachment anchors the disc during articulation, while the softer posterior superior attachment allows translation over the articular eminence

Matrix Development in Self-Assembly of Articular Cartilage

Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue.This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks.The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs

Tension-Compression Loading with Chemical Stimulation Results in Additive Increases to Functional Properties of Anatomic Meniscal Constructs

Objective: This study aimed to improve the functional properties of anatomically-shaped meniscus constructs through simultaneous tension and compression mechanical stimulation in conjunction with chemical stimulation. Methods: Scaffoldless meniscal constructs were subjected to simultaneous tension and compressive stimulation and chemical stimulation. The temporal aspect of mechanical loadingwas studied by employing two separate five day stimulation periods. Chemical stimulation consisted of the application of a catabolic GAG-depleting enzyme, chondroitinase ABC (C-ABC), and an anabolic growth factor, TGF-b1. Mechanical and chemical stimulation combinations were studied through a full-factorial experimental design and assessed for histological, biochemical, and biomechanical properties following 4 wks of culture. Results: Mechanical loading applied from days 10–14 resulted in significant increases in compressive, tensile, and biochemical properties of meniscal constructs. When mechanical and chemical stimuliwere combined significant additive increases in collagen per wet weight (4-fold), compressive instantaneous (3-fold) and relaxation (2-fold) moduli, and tensile moduli in the circumferential (4-fold) and radial (6-fold) directions were obtained. Conclusions: This study demonstrates that a stimulation regimen of simultaneous tension and compression mechanical stimulation, C-ABC, and TGF-b1 is able to create anatomic meniscus constructs replicating the compressive mechanica

Tension stimulation drives tissue formation in scaffold-free systems

Scaffold-free systems have emerged as viable approaches for engineering load-bearing tissues. However, the tensile properties of engineered tissues have remained far below the values for native tissue. Here, by using self-assembled articular cartilage as a model to examine the effects of intermittent and continuous tension stimulation on tissue formation, we show that the application of tension alone, or in combination with matrix remodelling and synthesis agents, leads to neocartilage with tensile properties approaching those of native tissue. Implantation of tension-stimulated tissues results in neotissues that are morphologically reminiscent of native cartilage. We also show that tension stimulation can be translated to a human cell source to generate anisotropic human neocartilage with enhanced tensile properties. Tension stimulation, which results in nearly sixfold improvements in tensile properties over unstimulated controls, may allow the engineering of mechanically robust biological replacements of native tissue

Cell-based tissue engineering strategies used in the clinical repair of articular cartilage

One of the most important issues facing cartilage tissue engineering is the inability to move technologies into the clinic. Despite the multitude of review articles on the paradigm of biomaterials, signals, and cells, it is reported that 90% of new drugs that advance past animal studies fail clinical trials (1). The intent of this review is to provide readers with an understanding of the scientific details of tissue engineered cartilage products that have demonstrated a certain level of efficacy in humans, so that newer technologies may be developed upon this foundation. Compared to existing treatments, such as microfracture or autologous chondrocyte implantation, a tissue engineered product can potentially provide more consistent clinical results in forming hyaline repair tissue and in filling the entirety of the defect. The various tissue engineering strategies (e.g., cell expansion, scaffold material, media formulations, biomimetic stimuli, etc.) used in forming these products, as collected from published literature, company websites, and relevant patents, are critically discussed. The authors note that many details about these products remain proprietary, not all information is made public, and that advancements to the products are continuously made. Nevertheless, by fully understanding the design and production processes of these emerging technologies, one can gain tremendous insight into how to best use them and also how to design the next generation of tissue engineered cartilage products