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This review details current efforts to tissue engineer the knee meniscus successfully. The meniscus is a fibrocartilaginous tissue found within the knee joint that is responsible for shock absorption, load transmission, and stability within the knee joint. If this tissue is damaged, either through tears or degenerative processes, then deterioration of the articular cartilage can occur. Unfortunately, there is a dearth in the amount of work done to tissue engineer the meniscus when compared to other musculoskeletal tissues, such as bone. This review gives a brief overview of meniscal anatomy, biochemical properties, biomechanical properties, and wound repair techniques. The discussion centers primarily on the different components of attempting to tissue engineer the meniscus, such as scaffold materials, growth factors, animal models, and culturing conditions. Our approach for tissue engineering the meniscus is also discussed.

The objective of this review is to provide basic information pertaining to biomechanical aspects of bone as they relate to tissue engineering. The review is written for the general tissue engineering reader, who may not have a biomechanical engineering background. To this end, biomechanical characteristics and properties of normal and repair cortical and cancellous bone are presented. Also, this chapter intends to describe basic structure-function relationships of these two types of bone. Special emphasis is placed on salient classical and modern testing methods, with both material and structural properties described.

Tissue engineering aims to create implantable biomaterials for the repair and regeneration of damaged tissues. In vitro tissue engineering is generally based on static culture, which limits access to nutrients and lacks mechanical signaling. Using shear stress is controversial because in some cases it can lead to cell death while in others it promotes tissue regeneration. To understand how shear stress works and how it may be used to improve neotissue function, a series of studies were performed. First, a tunable device was designed to determine optimal levels of shear stress for neotissue formation. Then, computational fluid dynamics modeling showed the device applies fluid-induced shear (FIS) stress spanning three orders of magnitude on tissue-engineered cartilage (neocartilage). A beneficial window of FIS stress was subsequently identified, resulting in up to 3.6-fold improvements in mechanical properties of neocartilage in vitro. In vivo, neocartilage matured as evidenced by the doubling of collagen content toward native values. Translation of FIS stress to human derived neocartilage was then demonstrated, yielding analogous improvements in mechanical properties, such as 168% increase in tensile modulus. To gain an understanding of the beneficial roles of FIS stress, a mechanistic study was performed revealing a mechanically gated complex on the primary cilia of chondrocytes that is activated by FIS stress. This series of studies places FIS stress into the arena as a meaningful mechanical stimulation strategy for creating robust and translatable neotissues, and demonstrates the ease of incorporating FIS stress in tissue culture.

Tissue engineers utilize a battery of expensive, time-consuming and destructive techniques to assess the composition and function of engineered tissues. A nondestructive solution to monitor tissue maturation would reduce costs and accelerate product development. As a first step toward this goal, two nondestructive, label-free optical techniques, namely multispectral fluorescent lifetime imaging (FLIm) and time-resolved fluorescence spectroscopy (TRFS), were investigated for their potential in evaluating the biochemical and mechanical properties of articular cartilage. Enzymatic treatments were utilized to selectively deplete cartilage of either collagen or proteoglycan, to produce a range of matrix compositions. Samples were assessed for their optical properties using a fiber-coupled optical system combining FLIm and TRFS, their biochemical and mechanical properties and by histological staining. Single and multivariable correlations were performed to evaluate relationships among these properties. FLIm- and TRFS-derived measurements are sensitive to changes in cartilage matrix and correlate with mechanical and biochemical assays. Mean fluorescence lifetime values extracted from FLIm images (375-410 nm spectral band) showed strong, specific correlations with collagen content (R2 = 0.79, p < 0.001) and tensile properties (R2 = 0.45, p = 0.02). TRFS lifetime measurements centered at 520 nm (with a 5 nm bandwidth) possessed strong, specific correlations with proteoglycan content (R2 = 0.59, p = 0.001) and compressive properties (R2 = 0.71, p < 0.001). Nondestructive optical assessment of articular cartilage, using a combination of FLIm- and TRFS-derived parameters, provided a quantitative method for determining tissue biochemical composition and mechanical function. These tools hold great potential for research, industrial and clinical settings.

ObjectiveThe application of cell-based therapies in regenerative medicine is hindered by the difficulty of acquiring adequate numbers of competent cells. For the knee meniscus in particular, this may be solved by harvesting tissue from neighboring tendons and ligaments. In this study, we have investigated the potential of cells from tendon and ligament, as compared to meniscus cells, to engineer scaffold-free self-assembling fibrocartilage.MethodSelf-assembling meniscus-shaped constructs engineered from a co-culture of articular chondrocytes and either meniscus, tendon, or ligament cells were cultured for 4 weeks with TGF-β1 in serum-free media. After culture, constructs were assessed for their mechanical properties, histological staining, gross appearance, and biochemical composition including cross-link content. Correlations were performed to evaluate relationships between biochemical content and mechanical properties.ResultsIn terms of mechanical properties as well as biochemical content, constructs engineered using tenocytes and ligament fibrocytes were found to be equivalent or superior to constructs engineered using meniscus cells. Furthermore, cross-link content was found to be correlated with engineered tissue tensile properties.ConclusionTenocytes and ligament fibrocytes represent viable cell sources for engineering meniscus fibrocartilage using the self-assembling process. Due to greater cross-link content, fibrocartilage engineered with tenocytes and ligament fibrocytes may maintain greater tensile properties than fibrocartilage engineered with meniscus cells.

Joint injury often leads to post-traumatic osteoarthritis (PTOA). Acute injury responses to trauma induce production of pro-inflammatory cytokines and catabolic enzymes, which promote chondrocyte apoptosis and degrade cartilage to potentiate PTOA development. Recent studies show that the rate-limiting step for transcriptional activation of injury response genes is controlled by cyclin-dependent kinase 9 (CDK9), and thus it is an attractive target for limiting the injury response. Here, we determined the effects of CDK9 inhibition in suppressing the injury response in mechanically-injured cartilage explants. Bovine cartilage explants were injured by a single compressive load of 30 % strain at 100 %/s, and then treated with the CDK9 inhibitor Flavopiridol. To assess acute injury responses, we measured the mRNA expression of pro-inflammatory cytokines, catabolic enzymes, and apoptotic genes by RT-PCR, and chondrocyte viability and apoptosis by TUNEL staining. For long-term outcome, cartilage matrix degradation was assessed by soluble glycosaminoglycan release, and by determining the mechanical properties with instantaneous and relaxation moduli. Our data showed CDK9 inhibitor markedly reduced injury-induced inflammatory cytokine and catabolic gene expression. CDK9 inhibitor also attenuated chondrocyte apoptosis and reduced cartilage matrix degradation. Lastly, the mechanical properties of the injured explants were preserved by CDK9 inhibitor. Our results provide a temporal profile connecting the chain of events from mechanical impact, acute injury responses, to the subsequent induction of chondrocyte apoptosis and cartilage matrix deterioration. Thus, CDK9 is a potential disease-modifying agent for injury response after knee trauma to prevent or delay PTOA development.

The ability to repair damaged cartilage is a major goal of musculoskeletal tissue engineering. Allogeneic (same species, different individual) or xenogeneic (different species) sources can provide an attractive source of chondrocytes for cartilage tissue engineering, since autologous (same individual) cells are scarce. Immune rejection of non-autologous hyaline articular cartilage has seldom been considered due to the popular notion of "cartilage immunoprivilege". The objective of this study was to determine the suitability of allogeneic and xenogeneic engineered neocartilage tissue for cartilage repair. To address this, scaffold-free tissue engineered articular cartilage of syngeneic (same genetic background), allogeneic, and xenogeneic origin were implanted into two different locations of the rabbit knee (n=3 per group/location). Xenogeneic engineered cartilage and control xenogeneic chondral explants provoked profound innate inflammatory and adaptive cellular responses, regardless of transplant location. Cytological quantification of immune cells showed that, while allogeneic neocartilage elicited an immune response in the patella, negligible responses were observed when implanted into the trochlea; instead the responses were comparable to microfracture-treated empty defect controls. Allogeneic neocartilage survived within the trochlea implant site and demonstrated graft integration into the underlying bone. In conclusion, the knee joint cartilage does not represent an immune privileged site, strongly rejecting xenogeneic but not allogeneic chondrocytes in a location-dependent fashion. This difference in location-dependent survival of allogeneic tissue may be associated with proximity to the synovium.Statement of significanceThrough a series of in vivo studies this research demonstrates that articular cartilage is not fully immunoprivileged. In addition, we now show that anatomical location of the defect, even within the same joint compartment, strongly influences the degree of the resultant immune response. This is one of the first investigations to show that (1) immune tolerance to allogeneic tissue engineered cartilage and (2) subsequent implant survival are dependent on the implant location and proximity to the synovium.

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use.

This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musculoskeletal cartilages—for example articular cartilage, meniscus, temporomandibular joint disc, and intervertebral disc—are characterized by low vascularity and cellularity, and are amenable to scaffold-free tissue engineering approaches. Scaffold-free approaches, particularly the self-assembling process, mimic elements of developmental processes underlying these tissues. Discussed are various scaffold-free approaches for musculoskeletal cartilage tissue engineering, such as cell sheet engineering, aggregation, and the self-assembling process, as well as the availability and variety of cells used. Immunological considerations are of particular importance as engineered tissues are frequently of allogeneic, if not xenogeneic, origin. Factors that enhance the matrix production and mechanical properties of these engineered cartilages are also reviewed, as the fabrication of biomimetically suitable tissues is necessary to replicate function and ensure graft survival in vivo. The concept of combining scaffold-free and scaffold-based tissue engineering methods to address clinical needs is also discussed. Inasmuch as scaffold-based musculoskeletal tissue engineering approaches have been employed as a paradigm to generate engineered cartilages with appropriate functional properties, scaffold-free approaches are emerging as promising elements of a translational pathway not only for musculoskeletal cartilages but for other tissues as well.

The purpose of this study was to determine suture-holding properties of tissue-engineered neocartilage relative to native articular cartilage. To this end, suture pull-out strength was quantified for native articular cartilage and for neocartilages possessing various mechanical properties. Suture-holding properties were examined in vitro and in vivo. Neocartilage from bovine chondrocytes was engineered using two sets of exogenous stimuli, resulting in neotissue of different biochemical compositions. Compressive and tensile properties and glycosaminoglycan, collagen, and pyridinoline cross-link contents were assayed (study 1). Suture pull-out strength was compared between neocartilage constructs, and bovine and leporine native cartilage. Uniaxial pull-out test until failure was performed after passing 6-0 Vicryl through each tissue (study 2). Subsequently, neocartilage was implanted into a rabbit model to examine short-term suture-holding ability in vivo (study 3). Neocartilage glycosaminoglycan and collagen content per wet weight reached 4.55 ± 1.62% and 4.21 ± 0.77%, respectively. Tensile properties for neocartilage constructs reached 2.6 ± 0.77% MPa for Young's modulus and 1.39 ± 0.63 MPa for ultimate tensile strength. Neocartilage reached ~ 33% of suture pull-out strength of native articular cartilage. Neocartilage cross-link content reached 50% of native values, and suture pull-out strength correlated positively with cross-link content (R² = 0.74). Neocartilage sutured into rabbit osteochondral defects was successfully maintained for 3 weeks. This study shows that pyridinoline cross-links in neocartilage may be vital in controlling suture pull-out strength. Neocartilage produced in vitro with one-third of native tissue pull-out strength appears sufficient for construct suturing and retention in vivo.

Osteoarthritis, a degenerative disease of the load-bearing joints, greatly reduces quality of life for millions of Americans and places a tremendous cost on the American healthcare system. Due to limitations of current treatments, tissue engineering of articular cartilage may provide a promising therapeutic option to treat cartilage defects. However, cartilage tissue engineering has yet to recapitulate the functional properties of native tissue. During normal joint loading, cartilage tissue experiences variations in osmolarity and subsequent changes in ionic concentrations. Motivated by these known variations in the cellular microenvironment, this study sought to improve the mechanical properties of neocartilage constructs via the application of hyperosmolarity and transient receptor potential vanilloid 4 (TRPV4) channel activator 4α-phorbol 12,13-didecanoate (4αPDD). It was shown that 4αPDD elicited significant increases in compressive properties. Importantly, when combined, 4αPDD positively interacted with hyperosmolarity to modulate its effects on tensile stiffness and collagen content. Thus, this study supports 4αPDD-activated channel TRPV4 as a purported mechanosensor and osmosensor that can facilitate the cell and tissue level responses to improve the mechanical properties of engineered cartilage. To our knowledge, this study is the first to systematically evaluate the roles of hyperosmolarity and 4αPDD on the functional (i.e., mechanical and biochemical) properties of self-assembled neotissue. Future work may combine 4αPDD-induced channel activation with other chemical and mechanical stimuli to create robust neocartilages suitable for treatment of articular cartilage defects.