Chondrocyte Hypertrophy in Osteoarthritis: Mechanistic Studies and Models for the Identification of New Therapeutic Strategies.

Articular cartilage shows limited self-healing ability owing to its low cellularity and avascularity. Untreated cartilage defects display an increased propensity to degenerate, leading to osteoarthritis (OA). During OA progression, articular chondrocytes are subjected to significant alterations in gene expression and phenotype, including a shift towards a hypertrophic-like state (with the expression of collagen type X, matrix metalloproteinases-13, and alkaline phosphatase) analogous to what eventuates during endochondral ossification. Present OA management strategies focus, however, exclusively on cartilage inflammation and degradation. A better understanding of the hypertrophic chondrocyte phenotype in OA might give new insights into its pathogenesis, suggesting potential disease-modifying therapeutic approaches. Recent developments in the field of cellular/molecular biology and tissue engineering proceeded in the direction of contrasting the onset of this hypertrophic phenotype, but knowledge gaps in the cause-effect of these processes are still present. In this review we will highlight the possible advantages and drawbacks of using this approach as a therapeutic strategy while focusing on the experimental models necessary for a better understanding of the phenomenon. Specifically, we will discuss in brief the cellular signaling pathways associated with the onset of a hypertrophic phenotype in chondrocytes during the progression of OA and will analyze in depth the advantages and disadvantages of various models that have been used to mimic it. Afterwards, we will present the strategies developed and proposed to impede chondrocyte hypertrophy and cartilage matrix mineralization/calcification. Finally, we will examine the future perspectives of OA therapeutic strategies.

Developmental biology-inspired tissue engineering by combining organoids and 3D bioprinting.

Very few tissue-engineered constructs could achieve the desired results in human clinical trials. The main reason is their inability to recapitulate the cellular conformation, biological, and mechanical functions of the native tissue. Here, we highlight the future avenues of tissue regeneration combining developmental biology, organoids, and 3D bioprinting. A deep mechanistic insight into the embryonic level and recapitulating them would be the most promising strategy in next-generation tissue engineering. Rather than focusing on the adult tissue features, the latest developmental re-engineering strategies replicate the developmental phases of tissue development. Integrating developmental re-engineering with 3D bioprinting can regulate several signaling pathways. This would further help to fabricate mini-organ constructs for transplantation or in vitro screening of drugs using an organ-on-a-chip platform.

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.

Blockage of bone morphogenetic protein signalling counteracts hypertrophy in a human osteoarthritic micro-cartilage model.

Bone morphogenetic protein (BMP) signalling plays a significant role during embryonic cartilage development and has been associated with osteoarthritis (OA) pathogenesis, being in both cases involved in triggering hypertrophy. Inspired by recent findings that BMP inhibition counteracts hypertrophic differentiation of human mesenchymal progenitors, we hypothesized that selective inhibition of BMP signalling would mitigate hypertrophic features in OA cartilage. First, a 3D in vitro OA micro-cartilage model was established using minimally expanded OA chondrocytes that was reproducibly able to capture OA-like hypertrophic features. BMP signalling was then restricted by means of two BMP receptor type I inhibitors, resulting in reduction of OA hypertrophic traits while maintaining synthesis of cartilage extracellular matrix. Our findings open potential pharmacological strategies for counteracting cartilage hypertrophy in OA and support the broader perspective that key signalling pathways known from developmental processes can guide the understanding, and possibly the mitigation, of adult pathological features.

Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells.

Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as ß-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.

Interplay between hereditary and environmental factors to establish an in vitro disease model of keratoconus.

Keratoconus (KC) is a bilateral corneal dystrophy and a multifactorial, multigenic disorder with an etiology involving a strong environmental component and complex inheritance patterns. The underlying pathophysiology of KC is poorly understood because of potential crosstalk between genetic-epigenetic variants possibly triggered by the environmental factors. Here, we decode the etiopathological basis of KC using genomic, transcriptomic, proteomic and metabolic approaches. The lack of relevant models that accurately imitate this condition has been particularly limiting in terms of the effective management of KC. Tissue-engineered in vitro models of KC could address this need and generate valuable insights into its etiopathology for the establishment of disease models to accelerate drug discovery.

Fleck-like deposits and swept source optical coherence tomography characteristics in a case of confirmed ocular chalcosis.

A 36-year-old male presented with history of injury in the left eye 3 years back with a copper wire. Examination revealed the presence of typical sunflower cataract with golden yellow deposits over the anterior lens capsule with dull glow and old vitreous hemorrhage. Non-contrast computerized tomography revealed retained intraocular foreign body in the pars plana region. The patient underwent phacoemulsification with intraocular lens implantation followed by pars plana vitrectomy and foreign body removal. Intraoperatively, fleck-like deposits were noted on the retinal surface in a circinate manner around the fovea and also over mid-peripheral retina. Postoperative swept source optical coherence tomography (SS-OCT) was performed to document the location of deposits and their characteristics. Limited literature exists regarding SS-OCT characteristics of ocular chalcosis.

Establishment of an in vitro organoid model of dermal papilla of human hair follicle.

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three-dimensional (3D) DP spheroids (DPS) with or without a silk-gelatin (SG) microenvironment, which showed enhanced DP-specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil-treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell-cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial-mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.

Silk-Based Bioinks for 3D Bioprinting.

3D bioprinting field is making remarkable progress; however, the development of critical sized engineered tissue construct is still a farfetched goal. Silk fibroin offers a promising choice for bioink material. Nature has imparted several unique structural features in silk protein to ensure spinnability by silkworms or spider. Researchers have modified the structure-property relationship by reverse engineering to further improve shear thinning behavior, high printability, cytocompatible gelation, and high structural fidelity. In this review, it is attempted to summarize the recent advancements made in the field of 3D bioprinting in context of two major sources of silk fibroin: silkworm silk and spider silk (native and recombinant). The challenges faced by current approaches in processing silk bioinks, cellular signaling pathways modulated by silk chemistry and secondary conformations, gaps in knowledge, and future directions acquired for pushing the field further toward clinic are further elaborated.

Regulation of fibrotic changes by the synergistic effects of cytokines, dimensionality and matrix: Towards the development of an in vitro human dermal hypertrophic scar model.

Current therapeutic strategies to reduce scarring in full thickness skin defect offer limited success due to poor understanding of scar tissue formation and the underlying signaling pathways. There is an urgent need to develop human cell based in vitro scar tissue models as animal testing is associated with ethical and logistic complications and inter-species variations. Pro-inflammatory cytokines play critical role in regulating scar development through complex interplay and interaction with the ECM and corresponding signaling pathways. In this context, we assessed the responses of cultured fibroblasts with respect to their differentiation into myofibroblasts using optimised cytokines (TGF-ß1, IL-6 and IL-8) for scar formation in 2D (tissue culture plate, collagen type I coated plate) vs 3D collagen type I gel based constructs. We attempted to deduce the role of dimensionality of cell culture matrix in modulating differentiation, function and phenotype of cultured fibroblasts. Validation of the developed model showed similarity to etiology and pathophysiology of in vivo hypertrophic scar with respect to several features: 1) transition of fibroblasts to myofibroblasts with convincing expression of α-SMA stress fibers; 2) contraction; 3) excessive collagen and fibronectin secretion; 4) expression of fibrotic ECM proteins (SPARC and Tenascin); 5) low MMP secretion. Most importantly, we elucidated the involvement of TGF-ß/SMAD and Wnt/ß-catenin pathways in developing in vitro dermal scar. Hence, this relatively simple in vitro human scar tissue equivalent may serve as an alternative for testing and designing of novel therapeutics and help in extending our understanding of the complex interplay of cytokines and related dermal scar specific signaling. STATEMENT OF SIGNIFICANCE: Scarring of the skin affects almost millions of people per year in the developed world alone, nevertheless the complex pathophysiology and the precise signaling mechanisms responsible for this phenomenon of skin scarring are still unknown. A number of anti-scar drugs are being developed and being tested on animals and monolayer models. However, testing the efficacy of these drugs on lab based 3D in vitro models may prove extremely useful in recapitulating the 3D microenvironment of the native scar tissue. In that context in this study we have demonstrated the development of 3D in vitro dermal scar model, by optimizing a constellation of factors, such as combination of cytokines (TGF-ß1,IL-6,IL-8) and cellular dimensionality in inducing the differentiation of dermal fibroblasts to myofibroblasts. This in vitro scar model was successful in replicating hallmark features of hypertrophic scar such as excessive synthesis of fibrotic extracellular matrix, perturbed matrix homeostasis, contraction, diminished MMP synthesis. The study also highlighted significant involvement of TGF-ß/SMAD and Wnt/ß-catenin signaling pathways in in vitro scar formation.

Differential Regulation of Hedgehog and Parathyroid Signaling in Mulberry and Nonmulberry Silk Fibroin Textile Braids.

Even after several decades of research, the most optimal source of silk for promoting osteogenesis in situ is still a subject of debate. A major gap in existing knowledge is role of underlying signaling mechanisms in both the mulberry and nonmulberry silk species that leads to the development of differential levels of osteogenesis. In our previous study, we elucidated the role of Wnt/ß-catenin signaling for promoting superior osteogenic differentiation in nonmulberry silk braids in the presence of TGF-ß and pro-osteogenic supplements. Here, we provide a comparative osteogenic analysis of the two most popular silk species (mulberry and nonmulberry silk), in the form of silk braids prepared from natively spun fibers, by conducting detailed gene expression profiling using 25 different osteogenic markers, followed by further validation by immunohistochemistry. Our study provides novel insights into the direct regulatory role of nonmulberry silk fibroin braids on hedgehog and parathyroid signaling pathways in controlling osteogenic differentiation of cultured human fetal osteoblasts (hFOBs), a phenomenon not very evident in the mulberry silk textile braids. Although both silk braids enabled adequate cellular attachment, proliferation, and extracellular collagen matrix formation, superior expression of osteogenic markers (ALP, VDR, Runx2), matrix proteins (Col1A2, OPN), and signaling molecules (GLI1, GLI2, Shh) with characteristic terminal osteocytic phenotype could only be observed in nonmulberry silk. Therefore, our study provided detailed insights into the development of engineered bone to be a prospective tissue equivalent with potential to provide the essential instructive elements for activating physiological pathways of bone differentiation. Such engineered constructs have potential for use as an in vitro model for drug testing and as scaffolds for bone regeneration strategies.

Developmental Biology-Inspired Strategies To Engineer 3D Bioprinted Bone Construct.

A major challenge in bone tissue engineering is to develop clinically conformant load-bearing bone constructs in a patient-specific manner. A paradigm shift would involve combination of developmental engineering and 3D bioprinting to optimize strategies focusing on close simulation of in vivo developmental processes using in vitro tissue engineering approaches. This study demonstrates that silk-gelatin bioink could activate the canonical Wnt/ß-catenin and Indian hedgehog (IHH) pathways during osteogenic differentiation of mesenchymal stem cells (TVA-BMSC), laden in 3D bioprinted constructs. Temporal gene expression related to early and terminal osteogenic differentiation of the TVA-BMSC in 3D bioprinted constructs closely followed the in vivo processes. This was evidenced by expression of early differentiation markers (RUNX2 and COL I), mid- and mid-to-late-stage markers (ALP, ON, OPN, and OCN), and terminal osteocytic genes (PDPN, DMP1, and SOST). Furthermore, a combinatorial effect of addition of T3 and simulation of the endochondral ossification route could activate the parathyroid hormone (PTH), IHH, and Wnt/ß-catenin pathways, thus improving the osteogenic differentiation potential of stem cells and improved mineralization. The endochondral ossification observed in vitro in our study shows stark similarities to in vivo endochondral ossification-based limb skeletal development, specifically (1) chondrogenic condensation and hypertrophic cartilaginous template development, (2) involvement of IHH signaling indicative of the development of bony collar by perichondral ossification, (3) involvement of Wnt/ß-catenin signaling, (4) involvement of PTH signaling, and (5) synthesis and deposition of bone-specific mineral. Thus, induction of differentiation of progenitor cells to osteoblasts in 3D bioprinted constructs, while recapitulating the developmental-biology-inspired endochondral ossification route, may offer an important therapeutic proposition to develop clinically conformant bone construct.

Establishment of in vitro model of corneal scar pathophysiology.

Corneal scarring is the major source of permanent blindness worldwide. The complex pathophysiology of corneal scarring is not comprehensibly understood as it involves the interaction of a constellation of pro-fibrotic cytokines influencing several signaling pathways involved in corneal scar development. In the present study, an attempt has been made to generate a relatively simple in vitro corneal scar model using primary corneal keratocytes by exogenously providing an optimized dose of combination of cytokines (TGF-ß1, IL-6, and IL-8) involved in scar formation in situ. Data obtained from gene and protein expression analysis depicted enhanced ECM production with discrete expression of myofibroblast specific markers. The protein-protein interactions associated these proteins to various pathways involved in wound healing, cellular migration, and cytoskeletal remodeling justifying high relevance to in vivo scar formation. Hence the developed model can be used to acquire understanding about corneal scar pathophysiology and thus might be useful for designing the treatment modalities and efficacies for controlling scar formation.

Role of chondroitin sulphate tethered silk scaffold in cartilaginous disc tissue regeneration.

Strategies for tissue engineering focus on scaffolds with tunable structure and morphology as well as optimum surface chemistry to simulate the anatomy and functionality of the target tissue. Silk fibroin has demonstrated its potential in supporting cartilaginous tissue formation both in vitro and in vivo. In this study, we investigate the role of controlled lamellar organization and chemical composition of biofunctionalized silk scaffolds in replicating the structural properties of the annulus region of an intervertebral disc using articular chondrocytes. Covalent attachment of chondroitin sulfate (CS) to silk is characterized. CS-conjugated silk constructs demonstrate enhanced cellular metabolic activity and chondrogenic redifferentiation potential with significantly improved mechanical properties over silk-only constructs. A matrix-assisted laser desorption ionization-time of flight analysis and protein-protein interaction studies help to generate insights into how CS conjugation can facilitate the production of disc associated matrix proteins, compared to a silk-only based construct. An in-depth understanding of the interplay between such extra cellular matrix associated proteins should help in designing more rational scaffolds for cartilaginous disc regeneration needs.