Articular Cartilage Engineering Research Articles

Comparison between articular chondrocytes and mesenchymal stromal cells for the production of articular cartilage implants.

Focal lesions of articular cartilage give rise to pain and reduced joint function and may, if left untreated, lead to osteoarthritis. Implantation of in vitro generated, scaffold-free autologous cartilage discs may represent the best treatment option. Here we compare articular chondrocytes (ACs) and bone marrow-derived mesenchymal stromal cells (MSCs) for their ability to make scaffold-free cartilage discs. Articular chondrocytes produced more extracellular matrix per seeded cell than mesenchymal stromal cells. Quantitative proteomics analysis showed that articular chondrocyte discs contained more articular cartilage proteins, while mesenchymal stromal cell discs had more proteins associated with cartilage hypertrophy and bone formation. Sequencing analysis revealed more microRNAs associated with normal cartilage in articular chondrocyte discs, and large-scale target predictions, performed for the first time for in vitro chondrogenesis, suggested that differential expression of microRNAs in the two disc types were important mechanisms behind differential synthesis of proteins. We conclude that articular chondrocytes should be preferred over mesenchymal stromal cells for tissue engineering of articular cartilage.

Nanofiber scaffolds based on extracellular matrix for articular cartilage engineering: A perspective.

Articular cartilage has a low self-repair capacity due to the lack of vessels and nerves. In recent times, nanofiber scaffolds have been widely used for this purpose. The optimum nanofiber scaffold should stimulate new tissue's growth and mimic the articular cartilage nature. Furthermore, the characteristics of the scaffold should match those of the cellular matrix components of the native tissue to best merge with the target tissue. Therefore, selective modification of prefabricated scaffolds based on the structure of the repaired tissues is commonly conducted to promote restoring the tissue. A thorough analysis is required to find out the architectural features of scaffolds that are essential to make the treatment successful. The current review aims to target this challenge. The article highlights different optimization approaches of nanofibrous scaffolds for improved cartilage tissue engineering. In this context, the influence of the architecture of nanoscaffolds on performance is discussed in detail. Finally, based on the gathered information, a future outlook is provided to catalyze development in this promising field.

Tissue Engineering of Articular Cartilage

Articular cartilage (AC) is the tissue that wraps moving joints in the body. As a tissue, it provides lubrication as well as load bearing in some joints. The tissue is subject to harsh chemical and mechanical environments in vivo and is characterized by being devoid of blood, nerve and lymph nodes. Upon injury, the tissue is incapable of healing. Damaged AC tissues mark the global disease of osteoarthritis (OA). As a pandemic, OA is prevalent worldwide and is ranked first for which patients seek treatments. Unfortunately, the disease has no current disease modifying drugs and it is only managed for symptomatic relief using pain killers, physical therapy and surgical procedures. In search for less invasive treatments, tissue engineering has been sought to provide the alternative. Tissue engineering refers to creation of eventually personalized tissues that can be used to replace damaged tissues in vitro. As an approach, it relies on seeding cells on scaffolds and incorporating the two in bioreactors that mimic the joint environment. Cells are then fed a medium with growth factors in it for a period of time until they form tissues. The tissues are then characterized to check if they represent the native tissue in structure and function. If not, the parameters used to engineer the tissue are revisited and the loop is repeated again. Here, tissue engineering of AC will be introduced. After that, the four pillars of tissue engineering (cells, scaffolds, growth factors, and bioreactors) will be discussed with questions that remain to be addressed. A section that discusses how engineered tissues are characterized will follow. Finally, the review ends with a summary of where the field is heading to realized quality AC tissues. This review is not meant to be comprehensive of existing literature.

Current Advances in the Regeneration of Degenerated Articular Cartilage: A Literature Review on Tissue Engineering and Its Recent Clinical Translation.

Functional ability is the basis of healthy aging. Articular cartilage degeneration is amongst the most prevalent degenerative conditions that cause adverse impacts on the quality of life; moreover, it represents a key predisposing factor to osteoarthritis (OA). Both the poor capacity of articular cartilage for self-repair and the unsatisfactory outcomes of available clinical interventions make innovative tissue engineering a promising therapeutic strategy for articular cartilage repair. Significant progress was made in this field; however, a marked heterogeneity in the applied biomaterials, biofabrication, and assessments is nowadays evident by the huge number of research studies published to date. Accordingly, this literature review assimilates the most recent advances in cell-based and cell-free tissue engineering of articular cartilage and also focuses on the assessments performed via various in vitro studies, ex vivo models, preclinical in vivo animal models, and clinical studies in order to provide a broad overview of the latest findings and clinical translation in the context of degenerated articular cartilage and OA.

Functional tissue engineering of articular cartilage for biological joint resurfacing-The 2021 Elizabeth Winston Lanier Kappa Delta Award.

Biological resurfacing of entire articular surfaces represents a challenging strategy for the treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. Here, we present the culmination of multiple avenues of interdisciplinary research leading to the development and testing of bioartificial cartilage for tissue-engineered resurfacing of the hip joint. The work is based on a novel three-dimensional weaving technology that is infiltrated with specific bioinductive materials and/or genetically-engineered stem cells. A variety of design approaches have been tested in vitro, showing biomimetic cartilage-like properties as well as the capability for long-term tunable and inducible drug delivery. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function. To date, these approaches have shown excellent preclinical success in a variety of animal studies, including the resurfacing of a large osteochondral defect in the canine hip, and are now well-poised for clinical translation.

3D-Printed Reinforcement Scaffolds with Targeted Biodegradation Properties for the Tissue Engineering of Articular Cartilage.

Achieving regeneration of articular cartilage is challenging due to the low healing capacity of the tissue. Appropriate selection of cell source, hydrogel, and scaffold materials are critical to obtain good integration and long-term stability of implants in native tissues. Specifically, biomechanical stability and in vivo integration can be improved if the rate of degradation of the scaffold material matches the stiffening of the sample by extracellular matrix secretion of the encapsulated cells. To this end, a novel 3D-printed lactide copolymer is presented as a reinforcement scaffold for an enzymatically crosslinked hyaluronic acid hydrogel. In this system, the biodegradable properties of the reinforced scaffold are matched to the matrix deposition of articular chondrocytes embedded in the hydrogel. The lactide reinforcement provides stability to the soft hydrogel in the early stages, allowing the composite to be directly implanted in vivo with no need for a preculture period. Compared to pure cellular hydrogels, maturation and matrix secretion remain unaffected by the reinforced scaffold. Furthermore, excellent biocompatibility and production of glycosaminoglycans and collagens are observed at all timepoints. Finally, in vivo subcutaneous implantation in nude mice shows cartilage-like tissue maturation, indicating the possibility for the use of these composite materials in one-step surgical procedures.

Preclinical Testing of New Hydrogel Materials for Cartilage Repair: Overcoming Fixation Issues in a Large Animal Model.

Reinforced hydrogels represent a promising strategy for tissue engineering of articular cartilage. They can recreate mechanical and biological characteristics of native articular cartilage and promote cartilage regeneration in combination with mesenchymal stromal cells. One of the limitations of in vivo models for testing the outcome of tissue engineering approaches is implant fixation. The high mechanical stress within the knee joint, as well as the concave and convex cartilage surfaces, makes fixation of reinforced hydrogel challenging. Methods. Different fixation methods for full-thickness chondral defects in minipigs such as fibrin glue, BioGlue®, covering, and direct suturing of nonenforced and enforced constructs were compared. Because of insufficient fixation in chondral defects, superficial osteochondral defects in the femoral trochlea, as well as the femoral condyle, were examined using press-fit fixation. Two different hydrogels (starPEG and PAGE) were compared by 3D-micro-CT (μCT) analysis as well as histological analysis. Results. Our results showed fixation of below 50% for all methods in chondral defects. A superficial osteochondral defect of 1 mm depth was necessary for long-term fixation of a polycaprolactone (PCL)-reinforced hydrogel construct. Press-fit fixation seems to be adapted for a reliable fixation of 95% without confounding effects of glue or suture material. Despite the good integration of our constructs, especially in the starPEG group, visible bone lysis was detected in micro-CT analysis. There was no significant difference between the two hydrogels (starPEG and PAGE) and empty control defects regarding regeneration tissue and cell integration. However, in the starPEG group, more cell-containing hydrogel fragments were found within the defect area. Conclusion. Press-fit fixation in a superficial osteochondral defect in the medial trochlear groove of adult minipigs is a promising fixation method for reinforced hydrogels. To avoid bone lysis, future approaches should focus on multilayered constructs recreating the zonal cartilage as well as the calcified cartilage and the subchondral bone plate.

Bio-inspired hydrogel composed of hyaluronic acid and alginate as a potential bioink for 3D bioprinting of articular cartilage engineering constructs

Bioprinting is a promising tool to fabricate well-organized cell-laden constructs for repair and regeneration of articular cartilage. The selection of a suitable bioink, in terms of composition and mechanical properties, is crucial for the development of viable cartilage substitutes. In this study, we focused on the use of one of the main cartilage components, hyaluronic acid (HA), to design and formulate a new bioink for cartilage tissue 3D bioprinting. Major characteristics required for this application such as printability, biocompatibility, and biodegradability were analyzed. To produce cartilage constructs with optimal mechanical properties, HA-based bioink was co-printed with polylactic acid (PLA). HA-based bioink was found to improve cell functionality by an increase in the expression of chondrogenic gene markers and specific matrix deposition and, therefore, tissue formation. These results indicate that it is a promising bioink candidate for cartilage tissue engineering based in 3D bioprinting. Statement of SignificanceThe recent appearance of 3D printing technology has enabled great advances in the treatment of osteochondral disorders by fabrication of cartilage tissue constructs that restore and/or regenerate damaged tissue. In this attempt, the selection of a suitable biomaterial, in terms of composition and mechanical properties, is crucial. In this study, we describe for first time the development of a bioink based on the main component of cartilage, HA, with suitable biological and mechanical properties, without involving toxic procedure, and its application in cartilage tissue bioprinting. Hybrid constructs prepared by co-printing this bioink and thermoplastic polymer PLA provided an optimal niche for chondrocyte growth and maintenance as well as mechanical properties necessary to support load forces exerted in native tissue. We highlight the translation potential of this HA-based bioink in the clinical arena.

Additive manufacturing of an elastic poly(ester)urethane for cartilage tissue engineering

Although a growing knowledge on the field of tissue engineering of articular cartilage exists, reconstruction or in-vitro growth of functional hyaline tissue still represents an unmet challenge. Despite the simplicity of the tissue in terms of cell population and absence of innervation and vascularization, the outstanding mechanical properties of articular cartilage, which are the result of the specificity of its extra cellular matrix (ECM), are difficult to mimic. Most importantly, controlling the differentiation state or phenotype of chondrocytes, which are responsible of the deposition of this specialized ECM, represents a milestone in the regeneration of native articular cartilage. In this study, we fabricated fused deposition modelled (FDM) scaffolds with different pore sizes and architectures from an elastic and biodegradable poly(ester)urethane (PEU) with mechanical properties that can be modulated by design, and that ranged the elasticity of articular cartilage. Cell culture in additive manufactured 3D scaffolds exceeded the chondrogenic potential of the gold-standard pellet culture. In-vitro cell culture studies demonstrated the intrinsic potential of elastic (PEU) to drive the re-differentiation of de-differentiated chondrocytes when cultured in-vitro, in differentiation or basal media, better than pellet cultures. The formation of neo-tissue was assessed as a high deposition of GAGs and fibrillar collagen II, and a high expression of typical chondrogenic markers. Moreover, the collagen II / collagen I ratio commonly used to evaluate the differentiation state of chondrocytes (ratio > 1 being chondrocytes and, ratio < 0 being de-differentiated chondrocytes) was higher than 5. Statement of significanceTissue engineering of articular cartilage requires material scaffolds capable of driving the deposition of a coherent and specific ECM representative of articular cartilage. Materials explored so far account for low mechanical properties (hydrogels), or are too stiff to mimic the elasticity of the native tissue (traditional polyesters). Here, we fabricated 3D fibrous scaffolds via FDM with a biodegradable poly(ester)urethane. The compressive Young`s modulus and elastic limit of the scaffolds can be tuned by designed, mimicking those of the native tissue. The designed scaffolds showed an intrinsic potential to drive the formation of a GAG and collagen II rich ECM, and to drive a stable chondrogenic cell phenotype.

Utilization of Finite Element Analysis for Articular Cartilage Tissue Engineering.

Scaffold design plays an essential role in tissue engineering of articular cartilage by providing the appropriate mechanical and biological environment for chondrocytes to proliferate and function. Optimization of scaffold design to generate tissue-engineered cartilage has traditionally been conducted using in-vitro and in-vivo models. Recent advances in computational analysis allow us to significantly decrease the time and cost of scaffold optimization using finite element analysis (FEA). FEA is an in-silico analysis technique that allows for scaffold design optimization by predicting mechanical responses of cells and scaffolds under applied loads. Finite element analyses can potentially mimic the morphology of cartilage using mesh elements (tetrahedral, hexahedral), material properties (elastic, hyperelastic, poroelastic, composite), physiological loads by applying loading conditions (static, dynamic), and constitutive stress–strain equations (linear, porous–elastic, biphasic). Furthermore, FEA can be applied to the study of the effects of dynamic loading, material properties cell differentiation, cell activity, scaffold structure optimization, and interstitial fluid flow, in isolated or combined multi-scale models. This review covers recent studies and trends in the use of FEA for cartilage tissue engineering and scaffold design.

Identifying chondrogenesis strategies for tissue engineering of articular cartilage

A key step in the tissue engineering of articular cartilage is the chondrogenic differentiation of mesenchymal stem cells (MSCs) into chondrocytes (native cartilage cells). Chondrogenesis is regulated by transforming growth factor-β (TGF-β), a short-lived cytokine whose effect is prolonged by storage in the extracellular matrix. Tissue engineering applications aim to maximise the yield of differentiated MSCs. Recent experiments involve seeding a hydrogel construct with a layer of MSCs lying below a layer of chondrocytes, stimulating the seeded cells in the construct from above with exogenous TGF-β and then culturing it in vitro. To investigate the efficacy of this strategy, we develop a mathematical model to describe the interactions between MSCs, chondrocytes and TGF-β. Using this model, we investigate the effect of varying the initial concentration of TGF-β, the initial densities of the MSCs and chondrocytes, and the relative depths of the two layers on the long-time composition of the tissue construct.

Chitosan scaffolds for cartilage regeneration: influence of different ionic crosslinkers on biomaterial properties

Chitosan is considered an elective scaffolding material in articular cartilage engineering due to its structural similarity with various Glycosamminogicans found in articular cartilage. Its poor mechanical properties, though, must be enhanced through a crosslinking treatment. Arginine (Arg) and tripolyphosphate (TPP), have been chosen as crosslinking agents, and their influence onto the chemico-physical properties (morphology, mechanical performances and stability behavior) of the resulting chitosan scaffold have been elucidated. The methodological conditions to produce a scaffold for the cartilage regeneration with tunable structural and chemical physical properties have been assessed. Furthermore, this study introduces Arginine as an innovative stabilizing agent.

Combination of Collagen-Based Scaffold and Bioactive Factors Induces Adipose-Derived Mesenchymal Stem Cells Chondrogenic Differentiation In vitro

Recently, multipotent mesenchymal stem cells (MSCs) have attracted much attention in the field of regenerative medicine due to their ability to give rise to different cell types, including chondrocytes. Damaged articular cartilage repair is one of the most challenging issues for regenerative medicine, due to the intrinsic limited capability of cartilage to heal because of its avascular nature. While surgical approaches like chondral autografts and allografts provide symptoms and function improvement only for a short period, MSC based stimulation therapies, like microfracture surgery or autologous matrix-induced chondrogenesis demonstrate to be more effective. The use of adult chondrocytes, which are the main cellular constituent of cartilage, in medical practice, is indeed limited due to their instability in monolayer culture and difficulty to collect donor tissue (articular and nasal cartilage). The most recent cartilage engineering approaches combine cells, biomaterial scaffold and bioactive factors to promote functional tissue replacements. Many recent evidences demonstrate that scaffolds providing specific microenvironmental conditions can promote MSCs differentiation toward a functional phenotype. In the present work, the chondrogenic potential of a new Collagen I based 3D scaffold has been assessed in vitro, in combination with human adipose-derived MSCs which possess a higher chondrogenic potential compared to MSCs isolated from other tissues. Our data indicate that the scaffold was able to promote the early stages of chondrogenic commitment and that supplementation of specific soluble factors was able to induce the complete differentiation of MSCs in chondrocytes as demonstrated by the appearance of cartilage distinctive markers (Sox 9, Aggrecan, Matrilin-1, and Collagen II), as well as by the cartilage-specific Alcian Blue staining and by the acquisition of typical cellular morphology. Such evidences suggest that the investigated scaffold formulation could be suitable for the production of medical devices that can be beneficial in the field of articular cartilage engineering, thus improving the efficacy and durability of the current therapeutic options.

Electrospun fibrous silk fibroin/poly(L-lactic acid) scaffold for cartilage tissue engineering.

For successful tissue engineering of articular cartilage, a scaffold with mechanical properties that match those of natural cartilage as closely as possible is needed. In the present study, we prepared a fibrous silk fibroin (SF)/poly(L-lactic acid) (PLLA) scaffold via electrospinning and investigated the morphological, mechanical, and degradation properties of the scaffolds fabricated using different electrospinning conditions, including collection distance, working voltage, and the SF:PLLA mass ratio. In addition, in vitro cell-scaffold interactions were evaluated in terms of chondrocyte adhesion to the scaffolds as well as the cytotoxicity and cytocompatibility of the scaffolds. The optimum electrospinning conditions for generating a fibrous SF/PLLA scaffold with the best surface morphology (ordered alignment and suitable diameter) and tensile strength (~1.5 MPa) were a collection distance of 20 cm, a working voltage of 15 kV, and a SF:PLLA mass ratio of S50P50. The degradation rate of the SF/PLLA scaffolds was found to be determined by the SF:PLLA mass ratio, and it could be increased by reducing the PLLA proportion. Furthermore, chondrocytes spread well on the fibrous SF/PLLA scaffolds and secreted extracellular matrix, indicating good adhesion to the scaffold. The cytotoxicity of SF/PLLA scaffold extract to chondrocytes over 24 and 48 h in culture was low, indicating that the SF/PLLA scaffolds are biocompatible. Chondrocytes grew well on the SF/PLLA scaffold after 1, 3, 5, and 7 days of direct contact, indicating the good cytocompatibility of the scaffold. These results demonstrate that the fibrous SF/PLLA scaffold represents a promising composite material for use in cartilage tissue engineering.

Characterization of Collagen Type I and II Blended Hydrogels for Articular Cartilage Tissue Engineering.

Biomaterials that provide signals present in the native extracellular matrix have been proposed as scaffolds to support improved cartilage regeneration. This study harnesses the biological activity of collagen type II and the superior mechanical properties of collagen type I by characterizing gels made of collagen type I and II blends. The collagen blend hydrogels were able to incorporate both types of collagen and retained chondroitin sulfate and hyaluronic acid. Cryo-scanning electron microscopy images showed that the 3:1 ratio of collagen type I to type II gels had a lower void space percentage (36.4%) than the 1:1 gels (46.5%). The complex modulus was larger for the 3:1 gels (G* = 5.0 Pa) compared to the 1:1 gels (G* = 1.2 Pa). The 3:1 blend consistently formed gels with superior mechanical properties compared to the other blends and has the potential to be implemented as a scaffold for articular cartilage engineering.

Modular Tissue Assembly Strategies for Biofabrication of Engineered Cartilage.

This review describes the prospects of applying modular assembly techniques and strategies for fabrication of advanced tissue engineered cartilage constructs. Articular cartilage is a tissue that has important functions in preserving and enabling locomotion. However, its limited intrinsic repair capacity and lack of current long-term clinical solutions makes it a candidate for repair or regeneration via tissue engineering strategies. Key advances in biofabrication and 3D bioprinting techniques allowing the specific placement of cells and tissues enable novel strategies to be adopted with increased chances of success. In particular, modular assembly, where separate biological components such as microtissue units, cellular building blocks or spheroids are combined with structural scaffold components to create a functional whole, offers potential as a new strategy for engineering of articular cartilage. Various modular assembly or bottom-up fabrication strategies have been investigated or applied for engineering of a range of tissues and cell types, however, modular approaches to cartilage engineering have been limited thus far. The integrative nature of many current approaches to engineering of articular cartilage means optimization of separate components (such as the scaffold and cells) is challenging, resulting in strategies which are less amenable to clinical scale-up or modification. In addition, current tissue engineering strategies may not replicate the function and complex structure of native tissue. This review outlines recent developments in fabrication of cellular or tissue modules as well as scaffold design where it impacts modular biofabrication, and discusses existing modular approaches applicable to articular cartilage regeneration and repair. Modular tissue assembly approaches allow complex hybrid constructs to be fabricated with direct control over both structural and cellular organization of pre-formed tissue units. The combination of modular assembly with automated biofabrication technologies may offer solutions to the development of optimal tissue-engineered cartilage constructs.

Gene Transfer and Gene Silencing in Stem Cells to Promote Chondrogenesis.

In stem cell-based chondrogenesis for articular cartilage regeneration, TGF-β3 is dosed to the stem cells to drive differentiation into chondrocytic cells. Meanwhile, type I collagen, which is endogenously expressed in some stem cells (e.g., synovium-derived mesenchymal stem cells) and upregulated by TGF-β3, poses a threat to chondrogenesis, as type I collagen may alter the components and stiffness of articular cartilage. Therefore, a wiser strategy would be to feed the cells with TGF-β3 while at the same time silencing the expression of type I collagen. In this chapter, methods for construction of adenoviral vectors and lentiviral vectors having both of the above functions are given. Their transduction into synovium-derived mesenchymal stem cells for articular cartilage engineering and following characterizations are also described.

Tissue engineering of articular cartilage: From bench to bed-side

Degeneration or defect of articular cartilage is a major predicament and if it is left untreated, it may lead to progressive damage and disability affecting every one disregard of their age. Although nonsurgical management of articular cartilage injury has remained largely the same over many years, surgical treatment keeps on evolving. Restorative techniques, mainly the cell-based therapies and autologous or allograft transplants continue to expand, giving surgeons more options for biologic reconstruction of the articular surfaces. Hence the field of articular cartilage tissue engineering which seeks to repair, restore and improve injured or diseased articular cartilage functionality has aroused deep interest and holds great potential for improving articular cartilage therapy. However despite this great evolution, therapeutic uncertainty in the restoration of damaged cartilage using tissue engineering approaches still remains unclear for the surgeon treating patients to make evidence-based decisions. This paper will give a general idea to different level of audiences in understanding the concept of tissue engineering from bench to bed-side regarding recent developments in this exciting field.

Engineering cartilage or endochondral bone: A comparison of different naturally derived hydrogels

Cartilaginous tissues engineered using mesenchymal stem cells (MSCs) have been shown to generate bone in vivo by executing an endochondral programme. This may hinder the use of MSCs for articular cartilage regeneration, but opens the possibility of using engineered cartilaginous tissues for large bone defect repair. Hydrogels may be an attractive tool in the scaling-up of such tissue engineered grafts for endochondral bone regeneration. In this study, we compared the capacity of different naturally derived hydrogels (alginate, chitosan and fibrin) to support chondrogenesis and hypertrophy of MSCs in vitro and endochondral ossification in vivo. In vitro, alginate and chitosan constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG), with chitosan constructs synthesizing the highest levels of collagen. Alginate and fibrin constructs supported the greatest degree of calcium accumulation, though only fibrin constructs calcified homogeneously. In vivo, chitosan constructs facilitated neither vascularization nor endochondral ossification, and also retained the greatest amount of sGAG, suggesting it to be a more suitable material for the engineering of articular cartilage. Both alginate and fibrin constructs facilitated vascularization and endochondral bone formation as well as the development of a bone marrow environment. Alginate constructs accumulated significantly more mineral and supported greater bone formation in central regions of the engineered tissue. In conclusion, this study demonstrates the capacity of chitosan hydrogels to promote and better maintain a chondrogenic phenotype in MSCs and highlights the potential of utilizing alginate hydrogels for MSC-based endochondral bone tissue engineering applications.

Promoting increased mechanical properties of tissue engineered neocartilage via the application of hyperosmolarity and 4α-phorbol 12,13-didecanoate (4αPDD)

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.