Native Cartilage Tissue Research Articles

Label-free 3D molecular imaging of living tissues using Raman spectral projection tomography

The ability to image tissues in three dimensions (3D) with label-free molecular contrast at the mesoscale would be a valuable capability in biology and biomedicine. Here, we introduce Raman spectral projection tomography (RSPT) for volumetric molecular imaging with optical sub-millimeter spatial resolution. We have developed a RSPT imaging instrument capable of providing 3D molecular contrast in transparent and semi-transparent samples. We also created a computational pipeline for multivariate reconstruction to extract label-free spatial molecular information from Raman projection data. Using these tools, we demonstrate imaging and visualization of phantoms of various complex shapes with label-free molecular contrast. Finally, we apply RSPT as a tool for imaging of molecular gradients and extracellular matrix heterogeneities in fixed and living tissue-engineered constructs and explanted native cartilage tissues. We show that there exists a favorable balance wherein employing Raman spectroscopy, with its advantages in live cell imaging and label-free molecular contrast, outweighs the reduction in imaging resolution and blurring caused by diffuse photon propagation. Thus, RSPT imaging opens new possibilities for label-free molecular monitoring of tissues.

Costal Chondrocyte–Derived Pellet-Type Autologous Chondrocyte Implantation Versus Microfracture for the Treatment of Articular Cartilage Defects: A 5-Year Follow-up of a Prospective Randomized Trial

Background: Costal chondrocyte–derived pellet-type autologous chondrocyte implantation (CCP-ACI) has been introduced as a new therapeutic option for the treatment of articular cartilage defects. We had previously conducted a randomized controlled trial comparing CCP-ACI versus microfracture at 1 year postoperatively. Purpose: To compare the efficacy and safety of CCP-ACI versus microfracture for the treatment of articular cartilage defects of the knee at 5 years postoperatively. Study Design: Randomized controlled trial; Level of evidence, 2. Methods: This study describes the mean 5-year follow-up of a previously published prospective clinical trial. The previous prospective trial compared the results of CCP-ACI versus microfracture until 1 year of follow-up. Of the 30 patients who were included in the previous study, 25 were followed up for 5 years. Patients were evaluated based on clinical outcome scores (Lysholm score, International Knee Documentation Committee score, Knee injury and Osteoarthritis Outcome Score [KOOS], and visual analog scale for pain), magnetic resonance imaging findings, and rates of treatment failure at last follow-up. Results: The MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) score in the CCP-ACI group was significantly higher than that in the microfracture group at 5 years (62.3 vs 26.7, respectively; P < .0001). The Lysholm score and KOOS score in the CCP-ACI group were significantly higher than those in the microfracture group at 5 years (84.5 vs 64.9, respectively, and 390.9 vs 303.0, respectively; P = .023 and P = .017, respectively). There was 1 case of treatment failure that occurred in the microfracture group. Conclusion: The present randomized controlled trial indicated that the results of both procedures clinically and statistically significantly improved at 1 and 5 years’ follow-up in treating cartilage defects, but the results of CCP-ACI were superior to those of microfracture. Magnetic resonance imaging conducted at 1 year and 5 years after CCP-ACI revealed statistically significant superior structural integration with native cartilage tissue compared with microfracture. Registration: NCT03545269 (ClinicalTrials.gov)

Anaphylatoxins and their corresponding receptors as potential drivers in cartilage calcification during osteoarthritis progression

ObjectiveThe complement cascade as major fluid phase innate immune system is activated during progression of osteoarthritis (OA). Generated anaphylatoxins and the corresponding receptors C3aR and C5aR1 are associated with the calcification of blood vessels and involved in osteogenic differentiation. This study aims on elucidating whether complement activation products contribute to cartilage calcification of OA cartilage. MethodHuman articular chondrocytes were osteogenically differentiated in vitro in the presence or absence of C3a, C5a, and bone morphogenetic protein (BMP) 2. Furthermore, macroscopically intact (OARSI grade ≤ 1) and highly degenerated human cartilage (OARSI grade ≥ 3) was used for C3aR and C5aR1 histochemistry. Calcification of the cartilage was assessed by Alizarin Red S and von Kossa staining. ResultsC3a and C5a amplified matrix mineralization during in vitro osteogenesis, while inhibition of the corresponding receptors impaired calcium deposition. Moreover, C3aR and C5aR1 expression was upregulated during osteogenic differentiation and also in degenerated cartilage. Additionally, anaphylatoxin receptor expression was positively associated with calcification of native cartilage tissue and calcium deposition during osteogenic differentiation. Finally, the pro-hypertrophic growth factor BMP2 induced the expression of C5aR1. ConclusionsOur findings indicate that anaphylatoxins and their receptors play a decisive role in cartilage calcification processes during OA progression.

Repair of full-thickness articular cartilage defects with a 3DP-anchored three-phase complex

IntroductionTo repair cartilage defect as well as the calcified cartilage layer (CCL) and bone tissue, there is need to fabricate a three-phase complex that mimics the natural cartilage tissue. Materials and methodsSF/Col-Ⅱ/HA scaffolds were constructed by low-temperature 3D printing, and to prepare a three-phase complex. The microstructure were showed using a SEM image analysis program. To observe collagen and glycosaminoglycan expression and analyze morphometric parameters, HE staining was performed to reveal new cartilage. Immunohistochemical were performed to investigate the collagen content and defect repair status in the new cartilage group in vitro and vivo. ResultsPhysical and biochemical properties and biocompatibility of three-phase complex met the requirements of constructing tissue-engineered cartilage. The OD values increased gradually at different time points. With increasing culture time, the OD values showed an upward trend. The HE and immunohistochemical staining results showed that new cartilage had formed at the defect and new cartilage formation occurred during in vivo repair. Conclusion3DP-anchored three-phase complexes have good physical and biochemical properties and biocompatibility and thus represent an alternative cartilage tissue engineering material.

Transplantable stem cell nanobridge scaffolds for accelerating articular cartilage regeneration

Microfracture technique for treating articular cartilage defects usually has poor clinical outcomes due to critical heterogeneity and extremely limited in quality. To improve the effects of current surgical technique (i.e., microfracture technique), we propose the transplantable stem cell nanobridge scaffold, acting as a protective bridge between host tissue and defected cartilage as well as microfracture-derived cells. Nanobridge scaffolds have a sophisticated nanoaligned structure with freestanding and flexible shapes for imposing direct structural guidance to cells including transplanted stem cells and host cells, and it can induce not only chondrocyte migration but also stem cell differentiation, maturation, and growth factor secretion. The transplantable stem cell nanobridge scaffold is capable of reconstructing the defected cartilage with homogeneous architecture and highly enhanced adhesive stress similar with native cartilage tissue by the synergistic effects of stem cells-based chondro-induction and nanotopography-based chondro-conduction. Our findings demonstrate a significant advancement in the traditional treatment technique by using a nanoengineered tool for achieving successful cartilage regeneration.

Quantitative T2 mapping monitoring the maturation of engineered elastic cartilage in a rabbit model

BackgroundCartilage tissue engineering provides a promising approach to reconstruct craniofacial defects, and a noninvasive method is needed to assess its effectiveness. Although magnetic resonance imaging (MRI) has been used to evaluate articular cartilage in vivo, few studies focused on its feasibility in monitoring engineered elastic cartilage (EC).MethodsAuricular cartilage, silk fibroin (SF) scaffold, and EC consisting of rabbit auricular chondrocytes and SF scaffold were transplanted subcutaneously into the rabbit back. In eight weeks after transplantation, grafts were imaged by MRI using PROSET, PDW VISTA SPAIR, 3D T2 VISTA, 2D MIXED T2 Multislice, and SAG TE multiecho sequences, followed by histological examination and biochemical analysis. Statistical analyses were performed to identify the association between T2 values and biochemical indicator values of EC.ResultsIn vivo imaging shows that 2D MIXED T2 Multislice sequence (T2 mapping) clearly distinguished the native cartilage, engineered cartilage and fibrous tissue. T2 values showed high correlations with cartilage-specific biochemical parameters at different time points, especially the elastic cartilage specific protein elastin (ELN, r= -0.939, P < 0.001).ConclusionQuantitative T2 mapping can effectively detect the in vivo maturity of engineered elastic cartilage after subcutaneously transplantation. This study would promote the clinical application of MRI T2 mapping in monitoring engineered elastic cartilage in the repair of craniofacial defects.

High-Speed-Microscopy for Scalable Quality Control in Automated Production of Stem Cell Spheroids for Tissue Engineering

The EU Horizon 2020 project »JointPromise« implies the conception and implementation of an end-to-end automated production platform for three-dimensional joint implants, paving the way for tissue-engineered implants able to regenerate deep osteochondral defects. Spheroid-based implants provide a novel approach in tissue engineering by aggregating progenitor cells into potent microtissues. After the differentiation of cartilaginous microtissues, functional joint implants are assembled via 3D bioprinting to match the complex structural organization of native cartilage tissue. As the automation approach of the project aims to overcome bottlenecks in manual production such as product variability, lack of scalability and high personnel costs, a high-throughput quality control system is crucial for the production of reliable Advanced Therapy Medicinal Products (ATMPs).By establishing not only a technical solution for the full digitization of the cell culture plates but also an intelligent image processing algorithm for the detection of the cell spheroids, relevant process parameters like size distribution and growth curves can be detected. Critical thresholds in spheroid growth are evaluated to minimize risks of carcinogenic tissue formation in vivo as well as to define harvest criteria to prevent inhomogeneous bioprinting results. In order to calculate the required throughput and elaborate optimization potentials of the automated spheroid production, voids in the cultivation vessel or disrupted aggregates due to media changes or transportation are detected. Ultimately, the highspeed-microscopy complies with the requirements of a high-throughput automated cell production platform to meet the rising demand for alternative therapeutic approaches in regenerative medicine.

Micromechanical property mismatch between pericellular and extracellular matrices regulates stem cell articular and hypertrophic chondrogenesis

<h2>Summary</h2> Within the complex microarchitecture of native cartilage tissue, the micromechanical properties of pericellular and extracellular matrices (PCM and ECM) potentially play important roles in developmental, physiological, and pathological processes. Here, we report a unique biomaterial-based engineering strategy to create cartilage-tissue equivalents possessing PCM-ECM microarchitecture of native cartilage, where human mesenchymal stem cell (hMSC)-laden soft microgels representing PCM are encapsulated in stiff hydrogels representing ECM. Mechanical property mismatches between soft PCM and stiff ECM under cyclic compression regulates hMSC proliferation and chondrogenesis. High PCM-ECM mechanical mismatch (softer PCM) and the presence of PCM degradation under cyclic compression individually or synergistically direct hMSC articular chondrogenesis through the proliferation-associated protein kinase C signaling pathway, whereas low PCM-ECM mechanical mismatch (stiffer PCM) is solely responsible for hMSC hypertrophic chondrogenesis through the yes-associated protein signaling pathway. Our findings highlight PCM-ECM mechanical property mismatch as a critical design cue under dynamic compression for hMSC-based cartilage repair.

A hybrid cartilage extracellular matrix-based hydrogel/poly (ε-caprolactone) scaffold incorporated with Kartogenin for cartilage tissue engineering.

Despite extensive studies, hydrogels are unable to meet the mechanical and biological requirements for successful outcomes in cartilage tissue engineering. In the present study, beta cyclodextrin (β-CD)-modified alginate/cartilage extracellular matrix (ECM)-based interpenetrating polymer network (IPN) hydrogel was developed for sustained release of Kartogenin (KGN). Furthermore, the hydrogel was incorporated within a 3D-printed poly (ε-caprolactone) (PCL)/starch microfiber network in order to reinforce the construct for cartilage tissue engineering. All the synthesized compounds were characterized by H1-NMR spectroscopy. The hydrogel/microfiber composite with a microfiber strand size and strand spacing of 300μm and 2mm, respectively showed a compressive modulus of 17.2MPa, resembling the properties of the native cartilage tissue. Considering water uptake capacity, degradation rate, mechanical property, cell cytotoxicity and glycosaminoglycan secretions, β-CD-modified hydrogel reinforced with printed PCL/starch microfibers with controlled release of KGN may be considered as a promising candidate for using in articular cartilage defects.

Preparation and Application of Decellularized ECM-Based Biological Scaffolds for Articular Cartilage Repair: A Review.

Cartilage regeneration is dependent on cellular-extracellular matrix (ECM) interactions. Natural ECM plays a role in mechanical and chemical cell signaling and promotes stem cell recruitment, differentiation and tissue regeneration in the absence of biological additives, including growth factors and peptides. To date, traditional tissue engineering methods by using natural and synthetic materials have not been able to replicate the physiological structure (biochemical composition and biomechanical properties) of natural cartilage. Techniques facilitating the repair and/or regeneration of articular cartilage pose a significant challenge for orthopedic surgeons. Whereas, little progress has been made in this field. In recent years, with advances in medicine, biochemistry and materials science, to meet the regenerative requirements of the heterogeneous and layered structure of native articular cartilage (AC) tissue, a series of tissue engineering scaffolds based on ECM materials have been developed. These scaffolds mimic the versatility of the native ECM in function, composition and dynamic properties and some of which are designed to improve cartilage regeneration. This review systematically investigates the following: the characteristics of cartilage ECM, repair mechanisms, decellularization method, source of ECM, and various ECM-based cartilage repair methods. In addition, the future development of ECM-based biomaterials is hypothesized.

Local depletion of proteoglycans mediates cartilage tissue repair in an ex vivo integration model

Successfully replacing damaged cartilage with tissue-engineered constructs requires integration with the host tissue and could benefit from leveraging the native tissue's intrinsic healing capacity; however, efforts are limited by a poor understanding of how cartilage repairs minor defects. Here, we investigated the conditions that foster natural cartilage tissue repair to identify strategies that might be exploited to enhance the integration of engineered/grafted cartilage with host tissue. We damaged porcine articular cartilage explants and using a combination of pulsed SILAC-based proteomics, ultrastructural imaging, and catabolic enzyme blocking strategies reveal that integration of damaged cartilage surfaces is not driven by neo-matrix synthesis, but rather local depletion of proteoglycans. ADAMTS4 expression and activity are upregulated in injured cartilage explants, but integration could be reduced by inhibiting metalloproteinase activity with TIMP3. These observations suggest that catabolic enzyme-mediated proteoglycan depletion likely allows existing collagen fibrils to undergo cross-linking, fibrillogenesis, or entanglement, driving integration. Catabolic enzymes are often considered pathophysiological markers of osteoarthritis. Our findings suggest that damage-induced upregulation of metalloproteinase activity may be a part of a healing response that tips towards tissue destruction under pathological conditions and in osteoarthritis, but could also be harnessed in tissue engineering strategies to mediate repair. Statement of significanceCartilage tissue engineering strategies require graft integration with the surrounding tissue; however, how the native tissue repairs minor injuries is poorly understood. We applied pulsed SILAC-based proteomics, ultrastructural imaging, and catabolic enzyme blocking strategies to a porcine cartilage explant model and found that integration of damaged cartilage surfaces is driven by catabolic enzyme-mediated local depletion of proteoglycans. Although catabolic enzymes have been implicated in cartilage destruction in osteoarthritis, our findings suggest that damage-induced upregulation of metalloproteinase activity may be a part of a healing response that tips towards tissue destruction under pathological conditions. They also suggest that this natural cartilage tissue repair process could be harnessed in tissue engineering strategies to enhance the integration of engineered cartilage with host tissue.

In Vitro and Ectopic In Vivo Studies toward the Utilization of Rapidly Isolated Human Nasal Chondrocytes for Single-Stage Arthroscopic Cartilage Regeneration Therapy.

Nasal chondrocytes (NCs) have a higher and more reproducible chondrogenic capacity than articular chondrocytes, and the engineered cartilage tissue they generate in vitro has been demonstrated to be safe in clinical applications. Here, we aimed at determining the feasibility for a single-stage application of NCs for cartilage regeneration under minimally invasive settings. In particular, we assessed whether NCs isolated using a short collagenase digestion protocol retain their potential to proliferate and chondro-differentiate within an injectable, swiftly cross-linked and matrix-metalloproteinase (MMP)-degradable polyethylene glycol (PEG) gel enriched with human platelet lysate (hPL). NC-hPL-PEG gels were additionally tested for their capacity to generate cartilage tissue in vivo and to integrate into cartilage/bone compartments of human osteochondral plugs upon ectopic subcutaneous implantation into nude mice. NCs isolated with a rapid protocol and embedded in PEG gels with hPL at low cell density were capable of efficiently proliferating and of generating tissue rich in glycosaminoglycans and collagen II. NC-hPL-PEG gels developed into hyaline-like cartilage tissues upon ectopic in vivo implantation and integrated with surrounding native cartilage and bone tissues. The delivery of NCs in PEG gels containing hPL is a feasible strategy for cartilage repair and now requires further validation in orthotopic in vivo models.

Comparison of Chondrogenesis Induction by TGF-β3, Kartogenin and Avocado/ soybean Unsaponifiables Between Laboratory and Animal Models

Background and Objectives Cartilage tissue engineering, by using stem cells, scaffolding and appropriate growth factors, seek to produce natural cartilage tissue to replace damaged tissue and solve the problems that exist in the treatment of cartilage damage. This study aims to compare chondrogenic differentiation of adipose-derived stem cells under the influence of the transforming growth factor beta 3 (TGF-β3), Kartogenin, and avocado/soybean unsaponifiables (ASU) on the fibrin scaffold in the laboratory and animal models. Subjects and Methods In this experimental laboratory study, after extraction of stem cells from human adipose tissue, induction of chondrogenic differentiation was done for 14 days on the fibrin scaffold in laboratory. The cells differentiated in the fibrin scaffold were transplanted subcutaneously under the skin of male rats for two weeks. Then, comparison of histological and immunohistochemical studies was performed in both laboratory and animal models. Results A significant increase in the density of Toluidine blue dye accumulation was observed in TGF-β3, Kartogenin and ASU groups in the animal model compared to the laboratory model. Immunohistochemical results for the collagen type X accumulation in the TGF-β3 group showed a significant increase in the animal model compared to the laboratory model. In the Kartogenin and ASU groups, the accumulation of collagen type Χ showed a significant decrease in the animal model compared to the laboratory model. Conclusion The implantation of differentiated cartilage cells in laboratory before subcutaneous transfer to the skin can help mature and complete the characteristics of the constructed cartilage.

Decellularized Avian Cartilage, a Promising Alternative for Human Cartilage Tissue Regeneration.

Articular cartilage defects, and subsequent degeneration, are prevalent and account for the poor quality of life of most elderly persons; they are also one of the main predisposing factors to osteoarthritis. Articular cartilage is an avascular tissue and, thus, has limited capacity for healing and self-repair. Damage to the articular cartilage by trauma or pathological causes is irreversible. Many approaches to repair cartilage have been attempted with some potential; however, there is no consensus on any ideal therapy. Tissue engineering holds promise as an approach to regenerate damaged cartilage. Since cell adhesion is a critical step in tissue engineering, providing a 3D microenvironment that recapitulates the cartilage tissue is vital to inducing cartilage regeneration. Decellularized materials have emerged as promising scaffolds for tissue engineering, since this procedure produces scaffolds from native tissues that possess structural and chemical natures that are mimetic of the extracellular matrix (ECM) of the native tissue. In this work, we present, for the first time, a study of decellularized scaffolds, produced from avian articular cartilage (extracted from Gallus Gallus domesticus), reseeded with human chondrocytes, and we demonstrate for the first time that human chondrocytes survived, proliferated and interacted with the scaffolds. Morphological studies of the decellularized scaffolds revealed an interconnected, porous architecture, ideal for cell growth. Mechanical characterization showed that the decellularized scaffolds registered stiffness comparable to the native cartilage tissues. Cell growth inhibition and immunocytochemical analyses showed that the decellularized scaffolds are suitable for cartilage regeneration.

Evaluation of the effects of glucosamine sulfate on poly(3- hydroxybutyrate) -chitosan/carbon nanotubes electrospun scaffold for cartilage tissue engineering applications

ABSTRACT Blend nanofibres composed of synthetic polymers with biological macromolecules, such as natural biopolymers, and 3D structures created via electrospinning technique, have a high potential for modification to promote cell growth and function. In this study, 5 and 7 wt% of glucosamine sulfate (GAS), as one of the key components in the extracellular matrix (ECM) of natural cartilage tissue, were added to poly (3-hydroxybutyrate)-chitosan (PHB-CS)/functionalized multiwalled carbon nanotubes (f-MWCNTs) solution (100:20:1) for production of electrospun scaffolds. Prepared fibrous scaffolds are characterized by SEM, FTIR, XRD, TGA, and DSC. Tensile tests are used to study their mechanical properties and their hydrophilicity is also assessed. subsequently, the drug release profile of the scaffolds, adipose stem cell proliferation, cell viability, and the differentiation of adipose stem cells to chondrocyte cells were evaluated for further cartilage tissue engineering application. Our results showed that the addition GAS has a positive effect on the hydrophilicity of the fibers and the initiation of chondrogenic differentiation. Without, effects on tensile strength of the scaffolds by adding 5%. So, that PHB-CS/f-MWCNTs scaffold containing GAS has more acceptable properties than the PHB or PHB-CS for cartilage tissue engineering.

Biochemical Aspects of Scaffolds for Cartilage Tissue Engineering; from Basic Science to Regenerative Medicine.

Chondral defects are frequent and important causes of pain and disability. Cartilage has limited self-repair and regeneration capacity. The ideal approach for articular cartilage defects is the regeneration of hyaline cartilage with sustainable symptom-free constructs. Tissue engineering provides new strategies for the regeneration of functional cartilage tissue through optimized scaffolds with architectural, mechanical, and biochemical properties similar to the native cartilage tissue. In this review, the basic science of cartilage structure, interactions between proteins, stem cells, as well as biomaterials, scaffold characteristics and fabrication methods, as well as current and potential therapies in regenerative medicine will be discussed mostly from a biochemical point of view. Furthermore, the recent trends in scaffold-based therapies and supplementary factors in cartilage tissue engineering will be considered.

An additive manufacturing-based 3D printed poly ɛ-caprolactone/alginate sulfate/extracellular matrix construct for nasal cartilage regeneration.

Various composite scaffolds with different fabrication techniques have been applied in cartilage tissue engineering. In this study, poly ɛ-caprolactone (PCL) was printed by fused deposition modeling method, and the prepared scaffold was filled with Alginate (Alg): Alginate-Sulfate (Alg-Sul) hydrogel to provide a better biomimetic environment and emulate the structure of glycosaminoglycans properly. Furthermore, to enhance chondrogenesis, different concentrations of decellularized extracellular matrix (dECM) were added to the hydrogel. For cellular analyses, the adipose-derived mesenchymal stem cells were seeded on the hydrogel and the results of MTT assay, live/dead staining, and SEM images revealed that the scaffold with 1% dECM had better viscosity, cell viability, and proliferation. The study was conducted on the optimized scaffold (1% dECM) to determine mechanical characteristics, chondrogenic differentiation, and results demonstrated that the scaffold showed mechanical similarity to the native nasal cartilage tissue along with possessing appropriate biochemical features, which makes this new formulation based on PCL/dECM/Alg:Alg-Sul a promising candidate for further in-vivo studies.

Additive manufacturing of cartilage-mimetic scaffolds as off-the-shelf implants for joint regeneration

Biomimetic scaffolds that provide a tissue-specific environment to cells are particularly promising for tissue engineering and regenerative medicine applications. The goal of this study was to integrate emerging additive manufacturing and biomaterial design strategies to produce articular cartilage (AC) mimetic scaffolds that could be used as ‘off-the-shelf’ implants for joint regeneration. To this end alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize porous alginate-based scaffolds and to support the sustained release of transforming growth factor-β3 (TGF-β3). Covalent crosslinking dramatically improved the elasticity of the alginate/alginate sulfate scaffolds, while scaffold architecture could be tailored using a directional freezing technique. Introducing such an anisotropic architecture was found to promote mesenchymal stem cell (MSC) infiltration into the scaffold and to direct the orientation of the deposited extracellular matrix, leading to the development of cartilage tissue with a biomimetic zonal architecture. In vitro experiments also demonstrated the capacity of the sulfated scaffolds to both enhance chondrogenesis of MSCs and to control the release of TGF-β3, leading to the development of a tissue rich in sGAG and type II collagen. The scaffolds were further reinforced with a 3D printed poly(lactide-co-ϵ-caprolactone) (PLCL) framework, leading to composite implants that were more elastic than those reinforced with polycaprolactone, and which better mimicked the bulk mechanical properties of native cartilage tissue. The ability of this composite scaffold to support chondrogenesis was then confirmed within a dynamic culture system. Altogether, these findings demonstrate the potential of such biomimetic scaffolds as putative ‘single-stage’ or ‘off-the-shelf’ strategies for AC regeneration.

The Effect of Neonatal, Juvenile, and Adult Donors on Rejuvenated Neocartilage Functional Properties.

Cartilage does not naturally heal, and cartilage lesions from trauma and wear-and-tear can lead to eventual osteoarthritis. To address long-term repair, tissue engineering of functional biologic implants to treat cartilage lesions is desirable, but the development of such implants is hindered by several limitations, including (1) donor tissue scarcity due to the presence of diseased tissues in joints, (2) dedifferentiation of chondrocytes during expansion, and (3) differences in functional output of cells dependent on donor age. Toward overcoming these challenges, (1) costal cartilage has been explored as a donor tissue, and (2) methods have been developed to rejuvenate the chondrogenic phenotype of passaged chondrocytes for generating self-assembled neocartilage. However, it remains unclear how the rejuvenation processes are influenced by donor age and, thus, how to develop strategies that specifically target age-related differences. Using histological, biochemical, proteomic, and mechanical assays, this study sought to determine the differences among neocartilage generated from neonatal, juvenile, and adult donors using the Yucatan minipig, a clinically relevant large animal model. Based on the literature, a relatively young adult population of animals was chosen due to a reduction in functional output of human articular chondrocytes after 40 years of age. After isolation, costal chondrocytes were expanded, rejuvenated, and self-assembled, and the neocartilages were assessed. The aggregate modulus values of neonatal constructs were at least 1.65-fold of those from the juvenile or adult constructs. Poisson's ratio also significantly differed among all groups, with neonatal constructs exhibiting values 49% higher than adult constructs. Surprisingly, other functional properties such as tensile modulus and glycosaminoglycan content did not significantly differ among groups. Total collagen content was slightly elevated in the adult constructs compared to neonatal and juvenile constructs. A more nuanced view using bottom-up mass spectrometry showed that Col2a1 protein was not significantly different among groups, but protein content of several other collagen subtypes (i.e., Col1a1, Col9a1, Col11a2, and Col12a1) was modulated by donor age. For example, Col12a1 protein content in adult constructs was found to be 102.9% higher than neonatal-derived constructs. Despite these differences, this study shows that different aged donors can be used to generate neocartilages of similar functional properties. Impact statement Tissue-engineered neocartilage can be generated with functional properties that mimic native cartilage tissue. However, cell sourcing challenges hinder clinical translation of tissue-engineered cartilage. Chondrocytes can be expanded and rejuvenated for the generation of functional self-assembled cartilage, making an allogeneic approach feasible. However, it is currently unclear if donor age impacts functional properties. In this study, using the Yucatan minipig as a clinically relevant large animal model, we demonstrate that functional properties of self-assembled neocartilage are relatively consistent regardless of donor age, suggesting that a wider range of donor ages may be used for cartilage tissue engineering than previously expected.

3D‐Printed Polyurethane Tissue‐Engineering Scaffold with Hierarchical Microcellular Foam Structure and Antibacterial Properties

This work proposes a facile fabrication strategy of polyurethane (TPU) tissue‐engineering scaffold with hierarchical structure, designed by combination of the fused deposition modeling (FDM) 3D printing and supercritical microcellular foaming by carbon dioxide (CO2). Further surface modification of TPU scaffold with graphene oxide (GO) was carried out by in situ polymerization of polydopamine (PDA) to ensure stable loading of GO with antibacterial properties. The influence of 3D‐printing temperature and speed on the microcellular cell morphology of TPU scaffold was investigated and optimized. The microcellular foaming and expansion process was indicated to compensate mechanical loss because of the gaps of stacking layers by FDM printing, and the reported tensile strength of 28 MPa matched well with that of natural cartilage tissue constructs. More interesting, the formation of interconnected open pores on the surface of foamed TPU scaffold shows much more enhanced cell adhesion because of the bigger specific surface area of microcellular structure. Similarly, the foamed TPU scaffold exhibits good absorption of GO nanosheets by the synthesized PDA on the surface and antibacterial properties, compared with the unfoamed TPU scaffold. This work offers a strategy for designing and manufacturing polymer tissue‐engineering scaffold with hierarchical structure and good antibacterial property.