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.

Integrating Melt Electrowriting and Fused Deposition Modeling to Fabricate Hybrid Scaffolds Supportive of Accelerated Bone Regeneration.

Emerging additive manufacturing (AM) strategies can enable the engineering of hierarchal scaffold structures for guiding tissue regeneration. Here, the advantages of two AM approaches, melt electrowriting (MEW) and fused deposition modelling (FDM), are leveraged and integrated to fabricate hybrid scaffolds for large bone defect healing. MEW is used to fabricate a microfibrous core to guide bone healing, while FDM is used to fabricate a stiff outer shell for mechanical support, with constructs being coated with pro-osteogenic calcium phosphate (CaP) nano-needles. Compared to MEW scaffolds alone, hybrid scaffolds prevent soft tissue collapse into the defect region and support increased vascularization and higher levels of new bone formation 12 weeks post-implantation. In an additional group, hybrid scaffolds are also functionalized with BMP2 via binding to the CaP coating, which further accelerates healing and facilitates the complete bridging of defects after 12 weeks. Histological analyses demonstrate that such scaffolds support the formation of well-defined annular bone, with an open medullary cavity, smooth periosteal surface, and no evidence of abnormal ectopic bone formation. These results demonstrate the potential of integrating different AM approaches for the development of regenerative biomaterials, and in particular, demonstrate the enhanced bone healing outcomes possible with hybrid MEW-FDM constructs.

Melt electrowritten scaffold architectures to mimic tissue mechanics and guide neo-tissue orientation.

All human tissues present with unique mechanical properties critical to their function. This is achieved in part through the specific architecture of the extracellular matrix (ECM) fibres within each tissue. An example of this is seen in the walls of the vasculature where each layer presents with a unique ECM orientation critical to its functions. Current adopted vascular grafts to bypass a stenosed/damaged vessel fail to recapitulate this unique mechanical behaviour, particularly in the case of small diameter vessels (<6 mm), leading to failure. Therefore, in this study, melt-electrowriting (MEW) was adopted to produce a range of fibrous scaffolds to mimic the extracellular matrix (ECM) architecture of the tunica media of the vasculature, in an attempt to match the mechanical and biological behaviour of the native porcine tissue. Initially, the range of collagen architectures within the native vessel was determined, and subsequently replicated using MEW (winding angles (WA) 45°, 26.5°, 18.4°, 11.3°). These scaffolds recapitulated the anisotropic, non-linear mechanical behaviour of native carotid blood vessels. Moreover, these grafts facilitated human mesenchymal stem cell (hMSC) infiltration, differentiation, and ECM deposition that was independent of WA. The bioinspired MEW fibre architecture promoted cell alignment and preferential neo-tissue orientation in a manner similar to that seen in native tissue, particularly for WA 18.4° and 11.3°, which is a mandatory requirement for long-term survival of the regenerated tissue post-scaffold degradation. Lastly, the WA 18.4° was translated to a tubular graft and was shown to mirror the mechanical behaviour of small diameter vessels within physiological strain. Taken together, this study demonstrates the capacity to use MEW to fabricate bioinspired scaffolds to mimic the tunica media of vessels and recapitulate vascular mechanics which could act as a framework for small diameter graft development to guide tissue regeneration and orientation.

Bioprinting of scaled-up meniscal grafts by spatially patterning phenotypically distinct meniscus progenitor cells within melt electrowritten scaffolds.

Meniscus injuries are a common problem in orthopedic medicine and are associated with a significantly increased risk of developing osteoarthritis. While developments have been made in the field of meniscus regeneration, the engineering of cell-laden constructs that mimic the complex structure, composition and biomechanics of the native tissue remains a significant challenge. This can be linked to the use of cells that are not phenotypically representative of the different zones of the meniscus, and an inability to direct the spatial organization of engineered meniscal tissues. In this study we investigated the potential of zone-specific meniscus progenitor cells (MPCs) to generate functional meniscal tissue following their deposition into melt electrowritten (MEW) scaffolds. We first confirmed that fibronectin selected MPCs from the inner and outer regions of the meniscus maintain their differentiation capacity with prolonged monolayer expansion, opening their use within advanced biofabrication strategies. By depositing MPCs within MEW scaffolds with elongated pore shapes, which functioned as physical boundaries to direct cell growth and extracellular matrix production, we were able to bioprint anisotropic fibrocartilaginous tissues with preferentially aligned collagen networks. Furthermore, by using MPCs isolated from the inner (iMPCs) and outer (oMPCs) zone of the meniscus, we were able to bioprint phenotypically distinct constructs mimicking aspects of the native tissue. An iterative MEW process was then implemented to print scaffolds with a similar wedged-shaped profile to that of the native meniscus, into which we deposited iMPCs and oMPCs in a spatially controlled manner. This process allowed us to engineer sulfated glycosaminoglycan and collagen rich constructs mimicking the geometry of the meniscus, with MPCs generating a more fibrocartilage-like tissue compared to the mesenchymal stromal/stem cells. Taken together, these results demonstrate how the convergence of emerging biofabrication platforms with tissue-specific progenitor cells can enable the engineering of complex tissues such as the meniscus.

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.

Bioprinting of structurally organized meniscal tissue within anisotropic melt electrowritten scaffolds.

The meniscus is characterised by an anisotropic collagen fibre network which is integral to its biomechanical functionality. The engineering of structurally organized meniscal grafts that mimic the anisotropy of the native tissue remains a significant challenge. In this study, inkjet bioprinting was used to deposit a cell-laden bioink into additively manufactured scaffolds of differing architectures to engineer fibrocartilage grafts with user defined collagen architectures. Polymeric scaffolds consisting of guiding fibre networks with varying aspect ratios (1:1; 1:4; 1:16) were produced using either fused deposition modelling (FDM) or melt electrowriting (MEW), resulting in scaffolds with different internal architectures and fibre diameters. Scaffold architecture was found to influence the spatial organization of the collagen network laid down by the jetted cells, with higher aspect ratios (1:4 and 1:16) supporting the formation of structurally anisotropic tissues. The MEW scaffolds supported the development of a fibrocartilaginous tissue with compressive mechanical properties similar to that of native meniscus, while the anisotropic tensile properties of these constructs could be tuned by altering the fibre network aspect ratio. This MEW framework was then used to generate scaffolds with spatially distinct fibre patterns, which in turn supported the development of heterogenous tissues consisting of isotropic and anisotropic collagen networks. Such bioprinted tissues could potentially form the basis of new treatment options for damaged and diseased meniscal tissue. STATEMENT OF SIGNIFICANCE: This study describes a multiple tool biofabrication strategy which enables the engineering of spatially organized fibrocartilage tissues. The architecture of MEW scaffolds can be tailored to not only modulate the directionality of the collagen fibres laid down by cells, but also to tune the anisotropic tensile mechanical properties of the resulting constructs, thereby enabling the engineering of biomimetic meniscal-like tissues. Furthermore, the inherent flexibility of MEW enables the development of zonally defined and potentially patient-specific implants.

Scaffold microarchitecture regulates angiogenesis and the regeneration of large bone defects.

Emerging 3D printing technologies can provide exquisite control over the external shape and internal architecture of scaffolds and tissue engineering (TE) constructs, enabling systematic studies to explore how geometric design features influence the regenerative process. Here we used fused deposition modelling (FDM) and melt electrowriting (MEW) to investigate how scaffold microarchitecture influences the healing of large bone defects. FDM was used to fabricate scaffolds with relatively large fibre diameters and low porosities, while MEW was used to fabricate scaffolds with smaller fibre diameters and higher porosities, with both scaffolds being designed to have comparable surface areas. Scaffold microarchitecture significantly influenced the healing response following implantation into critically sized femoral defects in rats, with the FDM scaffolds supporting the formation of larger bone spicules through its pores, while the MEW scaffolds supported the formation of a more round bone front during healing. After 12 weeksin vivo, both MEW and FDM scaffolds supported significantly higher levels of defect vascularisation compared to empty controls, while the MEW scaffolds supported higher levels of new bone formation. Somewhat surprisingly, this superior healing in the MEW group did not correlate with higher levels of angiogenesis, with the FDM scaffold supporting greater total vessel formation and the formation of larger vessels, while the MEW scaffold promoted the formation of a dense microvasculature with minimal evidence of larger vessels infiltrating the defect region. To conclude, the small fibre diameter, high porosity and high specific surface area of the MEW scaffold proved beneficial for osteogenesis and bone regeneration, demonstrating that changes in scaffold architecture enabled by this additive manufacturing technique can dramatically modulate angiogenesis and tissue regeneration without the need for complex exogenous growth factors. These results provide a valuable insight into the importance of 3D printed scaffold architecture when developing new bone TE strategies.

Tuning the Degradation Rate of Alginate-Based Bioinks for Bioprinting Functional Cartilage Tissue.

Negative foreign body responses following the in vivo implantation of bioprinted implants motivate the development of novel bioinks which can rapidly degrade with the formation of functional tissue, whilst still maintaining desired shapes post-printing. Here, we investigated the oxidation of alginate as a means to modify the degradation rate of alginate-based bioinks for cartilage tissue engineering applications. Raw and partially oxidized alginate (OA) were combined at different ratios (Alginate:OA at 100:0; 75:25; 50:50; 25:75; 0:100) to provide finer control over the rate of bioink degradation. These alginate blends were then combined with a temporary viscosity modifier (gelatin) to produce a range of degradable bioinks with rheological properties suitable for extrusion bioprinting. The rate of degradation was found to be highly dependent on the OA content of the bioink. Despite this high mass loss, the initially printed geometry was maintained throughout a 4 week in vitro culture period for all bioink blends except the 0:100 group. All bioink blends also supported robust chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs), resulting in the development of a hyaline-like tissue that was rich in type II collagen and negative for calcific deposits. Such tuneable inks offer numerous benefits to the field of 3D bioprinting, from providing space in a controllable manner for new extracellular matrix deposition, to alleviating concerns associated with a foreign body response to printed material inks in vivo.

Methacrylated Cartilage ECM-Based Hydrogels as Injectables and Bioinks for Cartilage Tissue Engineering.

Articular cartilage (AC) possesses a limited healing potential, meaning that untreated focal joint defects typically progress, leading to the development of degenerative diseases such as osteoarthritis. Several clinical strategies exist that aim to regenerate AC; however, recapitulation of a fully functional, load-bearing tissue remains a significant challenge. This can be attributed, at least in part, to a paucity of biomaterials that truly mimic the native tissue and provide appropriate cues to direct its regeneration. The main structural component of articular cartilage, type II collagen, does not readily gelate at body temperature, challenging the development of cartilage extracellular matrix (cECM)-derived injectable hydrogels and bioinks for AC tissue engineering and bioprinting applications. Here, we describe the development and rheological characterisation of a methacrylated cartilage ECM-based hydrogel/bioink (cECM-MA), which could be photocrosslinked when exposed to ultraviolet (UV) light. Functionalisation of the collagen backbone with methacryloyl groups had a negligible effect on triple helix stability, as demonstrated by circular dichroism spectroscopy. These cECM-MA bioinks demonstrated shear-thinning properties and could be loaded with bone marrow mesenchymal stem cells (BM-MSCs), micro-extruded to generate self-supporting 3D constructs of predefined size and shape, and then photocrosslinked using UV light. Analysis of the cell-laden constructs showed that the BM-MSCs were viable post-printing and underwent chondrogenesis in vitro, generating a tissue rich in sulphated glycosaminoglycans and collagens. These results support the use of methacrylated, tissue-specific ECM-derived hydrogels as bioinks for 3D bioprinting and/or as injectables for cartilage tissue engineering applications.

Biomanufacturing in low Earth orbit for regenerative medicine.

Research in low Earth orbit (LEO) has become more accessible. The 2020 Biomanufacturing in Space Symposium reviewed space-based regenerative medicine research and discussed leveraging LEO to advance biomanufacturing for regenerative medicine applications. The symposium identified areas where financial investments could stimulate advancements overcoming technical barriers. Opportunities in disease modeling, stem-cell-derived products, and biofabrication were highlighted. The symposium will initiate a roadmap to a sustainable market for regenerative medicine biomanufacturing in space. This perspective summarizes the 2020 Biomanufacturing in Space Symposium, highlights key biomanufacturing opportunities in LEO, and lays the framework for a roadmap to regenerative medicine biomanufacturing in space.

Targeted Type 2 Alveolar Cell Depletion. A Dynamic Functional Model for Lung Injury Repair.

Type 2 alveolar epithelial cells (AEC2) are regarded as the progenitor population of the alveolus responsible for injury repair and homeostatic maintenance. Depletion of this population is hypothesized to underlie various lung pathologies. Current models of lung injury rely on either uncontrolled, nonspecific destruction of alveolar epithelia or on targeted, nontitratable levels of fixed AEC2 ablation. We hypothesized that discrete levels of AEC2 ablation would trigger stereotypical and informative patterns of repair. To this end, we created a transgenic mouse model in which the surfactant protein-C promoter drives expression of a mutant SR39TK herpes simplex virus-1 thymidine kinase specifically in AEC2. Because of the sensitivity of SR39TK, low doses of ganciclovir can be administered to these animals to induce dose-dependent AEC2 depletion ranging from mild (50%) to lethal (82%) levels. We demonstrate that specific levels of AEC2 depletion cause altered expression patterns of apoptosis and repair proteins in surviving AEC2 as well as distinct changes in distal lung morphology, pulmonary function, collagen deposition, and expression of remodeling proteins in whole lung that persist for up to 60 days. We believe SPCTK mice demonstrate the utility of cell-specific expression of the SR39TK transgene for exerting fine control of target cell depletion. Our data demonstrate, for the first time, that specific levels of type 2 alveolar epithelial cell depletion produce characteristic injury repair outcomes. Most importantly, use of these mice will contribute to a better understanding of the role of AEC2 in the initiation of, and response to, lung injury.

Amniotic fluid stem cells inhibit the progression of bleomycin-induced pulmonary fibrosis via CCL2 modulation in bronchoalveolar lavage.

The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.

New ideas in lung regeneration: a personal view from Estoril.

10th ERS Lung Science Conference--rebuilding a diseased lung: repair and regeneration Estoril, Portugal, 30 March-1 April 2012 The 10th ERS Lung Science Conference, held in Estoril, Portugal, focused on rebuilding a diseased lung: repair and regeneration, seeking to understand, with some amount of precision, how the processes by which the vastly complex self-assembling, self-repairing machine that is a human lung actually develops from a few cells in the embryo, repairs itself or fails to and succumbs to disease. Thus, the major research themes focused on lung development, lung stem and progenitor cell populations, regenerative signaling mechanisms and tissue engineering and transplantation.

Modulating the alveolar milieu to enhance resolution of fibrotic lung injury.

Fibrotic lung injury is often attributed to a myriad of factors, including environmental exposure, age, genetic predisposition, epigenetics, coexisting conditions, acute lung injury, and viral infection. No effective therapies, other than lung transplantation, have proven effective against lung fibrosis. Loss of cellular homeostasis mechanisms in alveolar epithelial type I cells and any inability of type II progenitor cells to resist and repair epithelial injury are indicators that impaired response to injury and regeneration is a critical component of this disorder. The alveolar epithelium has a limited repertoire of responses to injury, which are dictated by the alveolar milieu, a repository of cytokines and growth factors that affect recruitment of other cells to the site of injury, or the proliferation of resident cells at the site of injury. The identification and characterization of the cytokines, growth factors, and other biomarkers that dictate the response to disease is key to understanding, diagnosing, treating, and determining the trajectory of various lung disorders. Corrective therapy of the alveolar milieu may therefore prove to be beneficial in many presently serious and incurable lung diseases that likely begin and progress with injury to the alveolar epithelium.

Cell-based therapies for lung disease.

INTRODUCTION OR BACKGROUND: The adult lung is a complex organ whose large surface area interfaces extensively with both the environment and circulatory system. Yet, in spite of the high potential for exposure to environmental or systemic harm, epithelial cell turnover in adult lung is comparatively slow. Moreover, loss of lung function with advancing age is becoming an increasingly costly healthcare problem. Cell-based therapies stimulating endogenous stem/progenitor cells or supplying exogenous ones have therefore become a prime translational goal. Alternatively when lung repair becomes impossible, replacement with tissue-engineered lung is an attractive emerging alternative using a decellularized matrix or bioengineered scaffold. SOURCES OF DATA: Endogenous and exogenous stem cells for lung therapy are being characterized by defining developmental lineages, surface marker expression, functions within the lung and responses to injury and disease. Seeding decellularized lung tissue or bioengineered matrices with various stem and progenitor cells is an approach that has already been used to replace bronchus and trachea in human patients and awaits further development for whole lung tissue. AREAS OF AGREEMENT: Cellular therapies have clear potential for respiratory disease. However, given the surface size and complexity of lung structure, the probability of a single cellular population sufficing to regenerate the entire organ, as in the bone marrow, remains low. Hence, lung regenerative medicine is currently focused around three aims: (i) to identify and stimulate resident cell populations that respond to injury or disease, (ii) to transplant exogenous cells which can ameliorate disease and (iii) to repopulate decellularized or bioengineered lung matrix creating a new implantable organ. AREAS OF CONTROVERSY: Lack of consensus on specific lineage markers for lung stem and progenitor cells in development and disease constrains transferability of research between laboratories and sources of cellular therapy. Furthermore, effectiveness of individual cellular therapies to correct gas exchange and provide other critical lung functions remains unproven. Finally, feasibility of autologous whole organ replacement has not been confirmed as a durable therapy. Growing points Cellular therapies for lung regeneration would be enhanced by better lineage tracing within the lung, the ability to direct differentiation of exogenous stem or progenitor cells, and the development of functional assays for cellular viability and regenerative properties. Whether endogenous or exogeneous cells will ultimately play a greater therapeutic role remains to be seen. Reducing the need for lung replacement via endogenous cell-mediated repair is a key goal. Thereafter, improving the potential of donor lungs in transplant recipients is a further area where cell-based therapies may be beneficial. Ultimately, lung replacement with autologous tissue-engineered lungs is another goal for cell-based therapy. Areas timely for developing research Defining 'lung stem or progenitor cell' populations in both animal models and human tissue may help. Additionally, standardizing assays for assessing the potential of endogenous or exogenous cells within the lung is important. Understanding cell-matrix interactions in real time and with biomechanical insight will be central for lung engineering. Cautionary note Communicating the real potential for cell-based lung therapy needs to remain realistic, given the keen expectations of patients with end-stage lung disease.