Chondroitinase ABC treatment improves the organization and mechanics of 3D bioprinted meniscal tissue

The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting. </p

Chondroitinase ABC treatment improves the organization and mechanics of 3D bioprinted meniscal tissue

The meniscus is a fibrocartilage tissue that is integral to the correct functioning of the knee joint. The tissue possesses a unique collagen fiber architecture that is integral to its biomechanical functionality. In particular, a network of circumferentially aligned collagen fibers function to bear the high tensile forces generated in the tissue during normal daily activities. The limited regenerative capacity of the meniscus has motivated increased interest in meniscus tissue engineering; however, the in vitro generation of structurally organized meniscal grafts with a collagen architecture mimetic of the native meniscus remains a significant challenge. Here we used melt electrowriting (MEW) to produce scaffolds with defined pore architectures to impose physical boundaries upon cell growth and extracellular matrix production. This enabled the bioprinting of anisotropic tissues with collagen fibers preferentially oriented parallel to the long axis of the scaffold pores. Furthermore, temporally removing glycosaminoglycans (sGAGs) during the early stages of in vitro tissue development using chondroitinase ABC (cABC) was found to positively impact collagen network maturation. Specially we found that temporal depletion of sGAGs is associated with an increase in collagen fiber diameter without any detrimental effect on the development of a meniscal tissue phenotype or subsequent extracellular matrix production. Moreover, temporal cABC treatment supported the development of engineered tissues with superior tensile mechanical properties compared to empty MEW scaffolds. These findings demonstrate the benefit of temporal enzymatic treatments when engineering structurally anisotropic tissues using emerging biofabrication technologies such as MEW and inkjet bioprinting. </p

Temporal enzymatic treatment to enhance the remodeling of multiple cartilage microtissues into a structurally organized tissue

Scaffold-free tissue engineering aims to recapitulate key aspects of normal developmental processes to generate biomimetic grafts. Although functional cartilaginous tissues are engineered using such approaches, considerable challenges remain. Herein, the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to using (millions of) individual cells to generate a cartilaginous graft are demonstrated. Key advantages include the generation of a richer extracellular matrix, more hyaline-like cartilage phenotype, and superior shape fidelity. A major drawback of aggregate engineering is that individual microtissues do not completely (re)model and remnants of their initial architectures remain throughout the macrotissue. To address this, a temporal enzymatic (chondroitinase-ABC) treatment is implemented to accelerate structural (re)modeling and shown to support robust fusion between adjacent microtissues, enhance microtissue (re)modeling, and enable the development of a more biomimetic tissue with a zonally organized collagen network. Additionally, enzymatic treatment is shown to modulate matrix composition, tissue phenotype, and to a lesser extent, tissue mechanics. This work demonstrates that microtissue self-organization is an effective method for engineering scaled-up cartilage grafts with a predefined geometry and near-native levels of matrix accumulation. Importantly, key limitations associated with using biological building blocks can be alleviated by temporal enzymatic treatment during graft development. </p

Temporal enzymatic treatment to enhance the remodeling of multiple cartilage microtissues into a structurally organized tissue

Scaffold-free tissue engineering aims to recapitulate key aspects of normal developmental processes to generate biomimetic grafts. Although functional cartilaginous tissues are engineered using such approaches, considerable challenges remain. Herein, the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to using (millions of) individual cells to generate a cartilaginous graft are demonstrated. Key advantages include the generation of a richer extracellular matrix, more hyaline-like cartilage phenotype, and superior shape fidelity. A major drawback of aggregate engineering is that individual microtissues do not completely (re)model and remnants of their initial architectures remain throughout the macrotissue. To address this, a temporal enzymatic (chondroitinase-ABC) treatment is implemented to accelerate structural (re)modeling and shown to support robust fusion between adjacent microtissues, enhance microtissue (re)modeling, and enable the development of a more biomimetic tissue with a zonally organized collagen network. Additionally, enzymatic treatment is shown to modulate matrix composition, tissue phenotype, and to a lesser extent, tissue mechanics. This work demonstrates that microtissue self-organization is an effective method for engineering scaled-up cartilage grafts with a predefined geometry and near-native levels of matrix accumulation. Importantly, key limitations associated with using biological building blocks can be alleviated by temporal enzymatic treatment during graft development. </p

Time-dependent anabolic response of hMSC-derived cartilage grafts to hydrostatic pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions. </p

Time-dependent anabolic response of hMSC-derived cartilage grafts to hydrostatic pressure

It is generally accepted that the application of hydrostatic pressure (HP) is beneficial for MSC chondrogenesis. There is, however, evidence to suggest that the timing of application might determine its impact on cell fate and tissue development. Furthermore, understanding how the maturity of engineered cartilage affects its response to the application of HP can provide critical insight into determining when such a graft is ready for in vivo implantation into a mechanically loaded environment. In this study, we systematically examined chondrogenic maturation of hMSCs over 35 days in the presence of TGF-β3 in vitro. At specific timepoints, the response of hMSCs to the application of HP following the removal of TGF-β3 was assessed; this partially models conditions such grafts will experience in vivo upon implantation. In free swelling culture, the expression of chondrogenic (COL2A1 and ACAN) and hypertrophic (COL10A1) markers increased with time. At early timepoints, the expression of such markers continued to increase following TGF-β3 withdrawal; however, this was not observed after prolonged periods of chondrogenic priming (35 days). Interestingly, the application of HP was only beneficial after 35 days of chondrogenic priming, where it enhanced sGAG synthesis and improved key chondrogenic gene ratios. It was also found that HP can facilitate a metabolic shift towards oxidative phosphorylation, which can be viewed as a hallmark of successfully differentiating MSCs. These results point to the importance of mechanical loading as a key stimulus for maintaining a chondrogenic phenotype once MSCs are removed from chemically defined culture conditions. </p

3D bioprinting of cartilaginous templates for large bone defect healing

Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. Statement of significance: Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-β3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds

Muticomponent melt-electrowritten vascular graft to mimic and guide regeneration of small diameter blood vessels

Cardiovascular disease is a leading cause of morbidity. Current treatments include vessel substitution using autologous/synthetic vascular grafts, but these commonly fail in small diameter applications, largely due to compliance mismatch and clot formation. In this study, a multicomponent vascular graft, that takes inspiration from native vessel architecture, is developed to overcome these limitations. Melt electrowriting (MEW) is used to produce tubular scaffolds with vascular-mimetic fiber architecture and mechanics, which is combined with a lyophilized fibrinogen matrix with tailored degradation kinetics to generate a hybrid graft. The MEW framework not only contributes to graft mechanics, but also provides contact guidance to direct cell/neotissue orientation and mimic the native tunica media. This construct is further functionalized with heparin, which in combination with the smooth extracellular matrix (ECM) surface, reduced platelet adhesion and clot formation providing a substrate for endothelization, thereby mimicking the function of the intima. Lastly, an outer electrospun layer representing the adventitia is added to improve elasticity and reduce permeability. This graft satisfies ISO implantability requirements, matches the compliance of native vessels, and reestablishes physiological flow with minimal clot formation in a preclinical model. Therefore, this graft represents an innovative off-the-shelf alternative to address the unmet clinical need for small-diameter vascular grafts. </p

3D printing of mechanically functional meniscal tissue equivalents using high concentration extracellular matrix inks

Decellularized extracellular matrix (dECM) has emerged as a promising biomaterial in the fields of tissue engineering and regenerative medicine due to its ability to provide specific biochemical and biophysical cues supportive of the regeneration of diverse tissue types. Such biomaterials have also been used to produce tissue-specific inks and bioinks for 3D printing applications. However, a major limitation associated with the use of such dECM materials is their poor mechanical properties, which limits their use in load-bearing applications such as meniscus regeneration. In this study, native porcine menisci were solubilized and decellularized using different methods to produce highly concentrated dECM inks of differing biochemical content and printability. All dECM inks displayed shear thinning and thixotropic properties, with increased viscosity and improved printability observed at higher pH levels, enabling the 3D printing of anatomically defined meniscal implants. With additional crosslinking of the dECM inks following thermal gelation at pH 11, it was possible to fabricate highly elastic meniscal tissue equivalents with compressive mechanical properties similar to the native tissue. These improved mechanical properties at higher pH correlated with the development of a denser network of smaller diameter collagen fibers. These constructs also displayed repeatable loading and unloading curves when subjected to long-term cyclic compression tests. Moreover, the printing of dECM inks at the appropriate pH promoted a preferential alignment of the collagen fibers. Altogether, these findings demonstrate the potential of 3D printing of highly concentrated meniscus dECM inks to produce mechanically functional and biocompatible implants for meniscal tissue regeneration. This approach could be applied to a wide variety of different biological tissues, enabling the 3D printing of tissue mimics with diverse applications from tissue engineering to surgical planning.</p

The influence of pH and salt concentration on the microstructure and mechanical properties of meniscus extracellular matrix-derived implants

Meniscus-related injuries are a common orthopedic challenge with an increasing incidence in the population. While the preservation of viable meniscal tissue is the preferred approach in repair strategies, complex or total traumatic lesions may require alternative therapeutic approaches such as meniscal reconstruction using allografts or engineered equivalents. Although clinical studies suggest promising outcomes with the use of acellular implants, further development is needed to improve their biological and mechanical requirements. Decellularized extracellular matrix (dECM) derived from menisci is a promising biomaterial for meniscus tissue engineering due to its recapitulation of the native tissue environment and the maintenance of tissue-specific cues. However, the associated mechanical limitations of dECM-derived scaffolds frequently impedes their adoption, requiring additional reinforcement or combining with stiffer biomaterials to increase their load-bearing properties. In this study, decellularized extracellular matrix was extracted and its fibrillation was controlled by adjusting both pH and salt concentrations to fabricate mechanically functional meniscal tissue equivalents. The effect of collagen fibrillation on the mechanical properties of the dECM constructs was assessed, and porcine-derived fibrochondrocytes were used to evaluate in vitro biocompatibility. It was also possible to fabricate meniscus-shaped implants by casting of the dECM and to render the implants suitable for off-the-shelf use by adopting a freeze-drying preservation method. Suture pull-out tests were also performed to assess the feasibility of using existing surgical methods to fix such implants within a damaged meniscus. This study highlights the potential of utilizing ECM-derived materials for meniscal tissue substitutes that closely mimic the mechanical and biological properties of native tissue. </p