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As an indispensable component of the extracellular matrix, perlecan (Pln) plays an essential role in cartilaginous tissue function. Although there exist studies suggesting that Pln expressed by cartilaginous tissues is critical for chondrogenesis, few papers have discussed the potential impact Pln may have on cartilage regeneration. In this review, we delineate Pln structure, biomechanical properties, and interactive ligands-which together contribute to the effect Pln has on cartilaginous tissue development. We also review how the signaling pathways of Pln affect cartilage development and scrutinize the potential application of Pln to divisions of cartilage regeneration, spanning vascularization, stem cell differentiation, and biomaterial improvement. The aim of this review is to deepen our understanding of the spatial and temporal interactions that occur between Pln and cartilaginous tissue and ultimately apply Pln in scaffold design to improve cell-based cartilage engineering and regeneration. STATEMENT OF SIGNIFICANCE: As a key component of the basement membrane, Pln plays a critical role in tissue development and repair. Recent findings suggest that Pln existing in the pericellular matrix surrounding mature chondrocytes is actively involved in cartilage regeneration and functionality. We propose that Pln is essential to developing an in vitro matrix niche within biological scaffolds for cartilage tissue engineering.

Relapsing polychondritis (RP) is a rare multisystem disorder characterized by inflammation and destruction of cartilaginous tissue. Atypical initial symptoms and signs may cause difficulties in the diagnosis of RP for pediatricians. In this article, we report a 9-year-old male who presented with stridor and hoarseness indicating upper respiratory tract involvement in the setting of chronic polyarthralgia. Flexible bronchoscopy revealed subglottic stenosis. Increasing awareness of early respiratory manifestations of RP will prevent life-threatening complications.

A major shortcoming in cartilage tissue engineering is the low biosynthetic response of chondrocytes. While different strategies have been investigated, a novel approach may be to control nutrient metabolism. Although known for their anaerobic metabolism, chondrocytes are more synthetically active under conditions that elicit mixed aerobic-anaerobic metabolism. Here, we postulate this metabolic switch induces HIF-1α signaling resulting in improved growth. Transition to different metabolic states can result in pooling of metabolites, several of which can stabilize HIF-1α by interfering with PHD2. Chondrocytes cultured under increased media availability accelerated tissue deposition with the greatest effect occurring at 2 mL/106 cells. Under higher media availability, metabolism switched from anaerobic to mixed aerobic-anaerobic. Around this transition, maximal changes in PHD2 activity, HIF-1α expression, and HIF-1 target gene expression were observed. Loss-of-function studies using YC-1 confirmed the involvement of HIF-1. Lastly, targeted metabolomic studies revealed that intracellular lactate and succinate correlated with PHD2 activity. This study demonstrates that cartilaginous tissue formation can be regulated by nutrient metabolism and that this response is mediated through changes in HIF-1α signalling. By harnessing this newly identified metabolic switch, engineered cartilage implants may be developed without the need for sophisticated methods which could aid translation to the clinic. This article is protected by copyright. All rights reserved.

Pankiv M. V., Paltov Ye. V., Masna Z. Z., Chelpanova I. V., Kovalska M. Ye. Structural features of the intervertebral disc in rats. Journal of Education, Health and Sport. 2021;11(07): 68-78. eISSN 2391-8306. DOI http://dx.doi.org/10.12775/JEHS.2021.11.07.006 https://apcz.umk.pl/czasopisma/index.php/JEHS/article/view/JEHS.2021.11.07.006 https://zenodo.org/record/5093282 The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. § 8. 2) and § 12. 1. 2) 22.02.2019. © The Authors 2021; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 14.06.2021. Revised: 25.06.2021. Accepted: 12.07.2021. UDK: 611.721.1.018.3.019 Structural features of the intervertebral disc in rats M. V. Pankiv, Ye. V. Paltov, Z. Z. Masna, I. V. Chelpanova, M. Ye. Kovalska Danylo Halytsky Lviv National Medical University, Lviv, Ukraine M.V. Pankiv, Ph. D-student at the Department of Normal Anatomy, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine; https://orcid.org/0000-0002-3714-2577 Ye.V. Paltov, Ph.D., Associate Professor, Head of the Laboratory of Electron Microscopy, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine; https://orcid.org/0000-0002-2622-4753 Z.Z. Masna, MD, Professor, Head of the Department of Operative Surgery and Topographical Anatomy, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine; https://orcid.org/0000-0003-2057-7061 I.V. Chelpanova, Ph.D., Associate Professor, Department of Histology, Cytology and Embryology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine https://orcid.org/ 0000-0001-5215-814X M. Ye. Kovalska Ph.D., Assistant of the department of pathological physiology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine; https://orcid.org/0000-0002-6232-3951 Abstract The aim of our work was to study the structural organization of the components of vertebral disc of rats on the microscopic level in the normal state. This goal was achieved by means of microscopic method of visualization of cellular components of the intervertebral disc of rats. Histological preparations were made in accordance with the conventional method of using the dye hematoxylin, eosin and azan according to the method of Heidenhain, PAS reaction according to MacManus and Alcian blue according to Steedman. As a result of the microstructural study, it was ascertained that the rat’s intervertebral disc normally consists of a gelatinous (pulpal) nucleus and a fibrous ring. It is separated from the vertebral bodies by a thin layer of cartilaginous tissue of the locking plates and hyaline cartilage, which covers the bodies of adjacent vertebrae. The data that rats do not have locking plates can be rarely found in the literature. The vessels verging the intervertebral disc radially -in front, back and from the sides, branch out from the vessels of vertebral periosteum. In the cartilaginous tissue of intervertebral disc, vessels are not visualized. Key words: intervertebral disc; structural components; norm; rat.

3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-β3 (TGF-β3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-β3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a 'single-stage' or 'point-of-care' strategy for regenerating cartilaginous tissues. STATEMENT OF SIGNIFICANCE: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrating networks in the bioink enables unique high-fidelity bioprinting and provides synergistic increases in mechanical properties. The presence of alginate sulfate enables the capacity of high affinity-binding of TGF-β3, which promoted robust chondrogenesis in vitro and in vivo.

The strain-generated potential (SGP) is a well-established mechanism in cartilaginous tissues whereby mechanical forces generate electrical potentials. In articular cartilage (AC) and the intervertebral disc (IVD), studies on the SGP have focused on fluid- and ionic-driven effects, namely Donnan, diffusion and streaming potentials. However, recent evidence has indicated a direct coupling between strain and electrical potential. Piezoelectricity is one such mechanism whereby deformation of most biological structures, like collagen, can directly generate an electrical potential. In this review, the SGP in AC and the IVD will be revisited in light of piezoelectricity and mechanotransduction. While the evidence base for physiologically significant piezoelectric responses in tissue is lacking, difficulties in quantifying the physiological response and imperfect measurement techniques may have underestimated the property. Hindering our understanding of the SGP further, numerical models to-date have negated ferroelectric effects in the SGP and have utilised classic Donnan theory that, as evidence argues, may be oversimplified. Moreover, changes in the SGP with degeneration due to an altered extracellular matrix (ECM) indicate that the significance of ionic-driven mechanisms may diminish relative to the piezoelectric response. The SGP, and these mechanisms behind it, are finally discussed in relation to the cell response.

Biomechanical changes to the collagen fibrillar architecture in articular cartilage are believed to play a crucial role in enabling normal joint function. However, experimentally there is little quantitative knowledge about the structural response of the Type II collagen fibrils in cartilage to cyclic loading in situ, and the mechanisms that drive the ability of cartilage to withstand long-term repetitive loading. Here we utilize synchrotron small-angle X-ray scattering (SAXS) combined with in-situ cyclic loading of bovine articular cartilage explants to measure the fibrillar response in deep zone articular cartilage, in terms of orientation, fibrillar strain and inter-fibrillar variability in healthy cartilage and cartilage degraded by exposure to the pro-inflammatory cytokine IL-1β. We demonstrate that under repeated cyclic loading the fibrils reversibly change the width of the fibrillar orientation distribution whilst maintaining a largely consistent average direction of orientation. Specifically, the effect on the fibrillar network is a 3-dimensional conical orientation broadening around the normal to the joint surface, inferred by 3D reconstruction of X-ray scattering peak intensity distributions from the 2D pattern. Further, at the intrafibrillar level, this effect is coupled with reversible reduction in fibrillar pre-strain under compression, alongside increase in the variability of fibrillar pre-strain. In IL-1β degraded cartilage, the collagen rearrangement under cyclic loading is disrupted and associated with reduced tissue stiffness. These finding have implications as to how changes in local collagen nanomechanics might drive disease progression or vice versa in conditions such as osteoarthritis and provides a pathway to a mechanistic understanding of such diseases. Statement of significance Structural deterioration in biomechanically loaded musculoskeletal organs, e.g., joint osteoarthritis and back pain, are linked to breakdown and changes in their collagen-rich cartilaginous tissue matrix. A critical component enabling cartilage biomechanics is the ultrastructural collagen fibrillar network in cartilage. However, experimental probes of the dynamic structural response of cartilage collagen to biomechanical loads are limited. Here, we use X-ray scattering during cyclic loading (as during walking) on joint tissue to show that cartilage fibrils resist loading by a reversible, three-dimensional orientation broadening and disordering mechanism at the molecular level, and that inflammation reduces this functionality. Our results will help understand how changes to small-scale tissue mechanisms are linked to ageing and osteoarthritic progression, and development of biomaterials for joint replacements., Graphical abstract Image, graphical abstract

Injuries and degenerative changes of tendons are common damages of the musculoskeletal system. Due to its hypovascular character the tendon has a limited natural ability to recover. For typical surgical treatment, the tendon integrity is restored, but in most cases, there occurs formation of the connective tissue scar resulting in structural and mechanical functionality disruption. The insufficient effectiveness of traditional therapy methods requires the search for alternative ways to restore damaged tendon tissues. This article discusses new effective methods for improving the treatment that base on the use of cellular technologies among which one of the main directions is mesenchymal stem cell application. Due to mesenchymal stem cells, there is a shift from pro-fibrotic and pro-inflammatory reactions of cells to pro-regenerative ones. Stem cells being multipotent and having among other things tenogenic potential are considered a promising material for repairing damaged tendons. The article also describes the sources of progenitor tendon cells including the tendon bundles and pericytes the main markers of which are Scx and Mkx that are proteins of the transcription factor superfamily, and Tnmd that is transmembrane glycoprotein.The growth factors that not only enhance the proliferative activity of mesenchymal stem cells but also promote in vitro tenogenic genes expression as well as the collagen Itype production what is necessary for tendon formation are considered. Along with growth factors, the morphogenetic protein BMP14 is presented, this protein increases themesenchymal stem cell proliferation and contributes directed tenogenic differentiation of these cells, suppressing their adipogenic and chondrogenic potentials.In recent years, mesenchymal stem cells have been used both separately and in combination with various growth factors and different three-dimensional structures providing the interaction with all of the cell types.The issues of the latest 3D-bioprinting technology allowing to make tissue-like structures for replacement damaged tissues and organs are discussed. 3D-bioprinting technology is known to allow acting exact spatio-temporal control of the distribution of cells, growth factors, small molecules, drugs and biologically active substances.

The results of experimental work on modeling deforming osteoarthrosis of the knee joint in small rodents are presented. The pathogenetic basis of this process is a violation of the blood supply and the distribution of physiological load in the joint. Variants of rigid and non-rigid limb immobilization systems are proposed. The results of morphofunctional assessment are given, indicating a violation of the anatomical and histological structure of the metaphysis, pineal gland and metaepiphysis of the femur and tibia. The leading role in the development of deforming osteoarthritis of the knee belongs to the violation of the physiological distribution of the dynamic load on the joint, and the most vulnerable to the progression of this process are subchondral structures. Violation of regional blood circulation of the lower limb of the animal was achieved by crossing the femoral artery. Limb immobilization was carried out for 4 weeks. The results were evaluated by the severity of changes in the morphological structure of the operated joints and radiographic examination. The high mitotic activity of cellular elements, revealed as a result of histological examination in all groups of animals, indicates the rapid formation of vascular collaterals in rats. It was noted that with non-rigid immobilization of the joint with a plaster cast, the cartilaginous tissue of the articular surface becomes thinner, while the subchondral structures change slightly. With severe immobilization of the joint in the external fixation apparatus, significant changes are revealed precisely in the structure of subchondral structures. In animals subjected to a two-week rehabilitation period after dismantling the external fixation apparatus, a further progression of the pathological process was noted. Thus, with severe immobilization of the limb in the experiment, it was possible to obtain a stable joint deformation corresponding to the 3rd clinical and radiological stage of osteoarthritis.

Background. Charcot arthropathy is a serious medical and social problem. Histological studies of foot joints components in Charcot arthropathy complicated by osteomyelitis are few. The purpose of this study was to assess structural changes in the articular cartilage and subchondral bone of the foot joints in Charcot arthropathy complicated by osteomyelitis. Materials and Methods. The bone-cartilage fragments of the ankle, subtalar and metatarsophalangeal joints with the surrounding soft tissues of 20 patients with Charcot arthropathy complicated by chronic osteomyelitis were examined. Part of the material was fixed in neutral formalin. All samples were subjected to standard histological processing. Paraffin sections were stained by Masson’s tricolor method, hematoxylin and eosin. Part of the material was embedded in epoxy resins. Then semi-thin sections were stained with methylene blue and basic fuchsin. Histological preparations were studied by digitizing images under the AxioScope A1 microscope (Carl Zeiss MicroImaging GmbH, Germany).The phase of chronic osteomyelitis inflammation was assessed semi-quantitatively using the histopathological scale by A. Tiemann et al. (2014). Results. In 80% of the patients, the inflammatory phase of chronic osteomyelitis was characterized as active and subacute. In all cases, the areas with full-layer of articular cartilage unweaving, up to the subchondral zone, with cartilaginous tissue fragments rejection into the joint cavity were revealed. Cytoarchitectonics was disrupted. The main part of chondrocytes was in a state of destruction. The articular surface was covered with pannus. There were no basophilic line and the zone of calcified cartilage. The hyaline cartilage was replaced by granulation and/or fibrous tissue. An inflammatory infiltrates was noted in the superficial and deep areas of the cartilage. The impairment of the structure and/or complete absence of the subchondral bone due to the high activity of osteoclasts in the subchondral zone were revealed. An excessive amount of osteoclasts at the border with the articular cartilage was noted, while the signs of reparative bone formation were poorly expressed. Edema and thickening of the vascular walls of the microvasculature were recorded. Conclusion. The microscopic examination of the foot joints in Charcot arthropathy complicated by osteomyelitis revealed structural impairment and/or complete absence of the subchondral bone due to the high activity of osteoclasts in the subchondral zone. Structural changes in the subchondral bone and synovial pannus led to irreversible destruction of articular cartilage and the penetration of infection. These should be taken into account in surgical planning.

Summary Objective Sprifermin (recombinant human fibroblast growth factor-18), a potential disease-modifying osteoarthritis (OA) drug, demonstrated dose-dependent effects on femorotibial cartilage thickness (by quantitative magnetic resonance imaging [MRI]) in the phase II FORWARD study. This post-hoc analysis evaluated the potential effects of sprifermin on several articular structures in the whole joint over 24 months using semi-quantitative MRI assessment. Design Patients aged 40–85 years with symptomatic radiographic knee OA, Kellgren–Lawrence grade 2 or 3, and medial minimum joint space width ≥2.5 mm in the target knee were randomized (1:1:1:1:1) to receive three double-blinded, once-weekly, intra-articular injections of sprifermin 30 μg or 100 μg or placebo every 6 (q6mo) or 12 months. 1.5- or 3 T MRIs were read using the Whole-Organ Magnetic Resonance Imaging Score (WORMS) system at baseline and 24 months. Change from baseline at 24 months on compartment and/or whole knee level was assessed for cartilage morphology, bone marrow lesions (BMLs), and osteophytes by delta-subregional and delta-sum (DSM) approaches. Menisci, Hoffa-synovitis, and effusion-synovitis were also evaluated for worsening. Results 549 patients were included. Dose-dependent treatment effects from baseline to 24 months were observed on cartilage morphology (sprifermin 100 μg q6mo vs placebo; mean DSM (95% confidence interval [CI]) −0.6 (−1.5, 0.2); less cartilage worsening) in the entire knee and BMLs sprifermin 100 μg q6mo vs placebo; mean DSM (95% CI) −0.2 (−0.5, 0.1) in the patellofemoral compartment. No effects over 24 months were observed on osteophytes, menisci, Hoffa-synovitis or effusion-synovitis. Conclusions Positive effects associated with sprifermin were observed for cartilage morphology changes, and BML improvement. There were no meaningful negative or positive effects associated with sprifermin in the other joint tissues examined.

Objectives Residual hip dysplasia is the most common underlying condition leading to secondary osteoarthritis (OA) of the hip. Subchondral bone alterations in OA secondary to hip dysplasia (HD-OA) are poorly investigated. The aim of the present study was to analyse the microarchitecture, bone remodelling and pathological alterations of subchondral bone in femoral heads from patients with HD-OA. Methods Subchondral bone specimens were extracted from both weight-bearing and non–weight-bearing regions of femoral heads from 20 patients with HD-OA and 20 patients with osteoporotic femoral neck fracture, during hip replacement surgery. Micro-CT and histological examination were performed to assess the microarchitecture and histopathological changes. Results The weight-bearing subchondral bone showed significantly more sclerotic microarchitecture and higher bone remodelling level in HD-OA as compared with osteoporosis. In the non–weight-bearing region, the two diseases shared similar microarchitectural characteristics, but higher bone remodelling level was detected in HD-OA. Distinct regional differences were observed in HD-OA, whereas the two regions exhibited similar characteristics in osteoporosis. In addition, HD-OA displayed more serious pathological alterations, including subchondral bone cyst, metaplastic cartilaginous tissue, bone marrow oedema and fibrous tissue, especially in the weight-bearing region. Conclusions Osteoarthritic deteriorations of subchondral bone induced by hip dysplasia spread throughout the whole joint, but exhibit region-dependent variations, with the weight-bearing region more seriously affected. Biomechanical stress might exert a pivotal impact on subchondral bone homeostasis in hip dysplasia. The translational potential of this article The histomorphometric findings in the project indicate an early intervention for the development of hip dysplasia in clinic.

Injectable hydrogels have attracted increasing attention because of convenient clinical operation, non-invasive surgical procedure and seamless filling of irregular defects. Here, injectable di-self-crosslinking HSMSSA hydrogel was formed via fast thiol/maleimide click chemistry reaction and thiol oxidation reaction as primary and secondary self-crosslinking network, respectively. Molecular weight and precursor concentration significantly affected physichemical properties and biological functions of hydrogels. Although single HSMSSA gel (0.1 M Da, 10 mg/mL) had moderate injectability, preferable mechanical properties and good proliferative ability of chondrocytes in vitro, and could greatly promote cartilaginous tissue formation in vivo, the lack of adhesion sites resulted in an untenable situation in maintaining effective connections among newborn cell clusters. However, the biomimetic injectable di-self-crosslinking blend hydrogel by combing injectable HSMSSA and bioactive Col I had improved resistance to degradation, chondrocytes adhesion and proliferation, especially for multiples ascending genes expression level associated with hyaline cartilage formation and polyproteoglycan secretion, which might be a potential clinical treatment strategy for constructing injectable cartilage repair filler by combining expanded autologous chondrocytes. Statement of Significance An injectable di-self-crosslinking Hyaluronan–Based hydrogel was formed via fast thiol/maleimide click chemistry reaction and thiol oxidation reaction as primary/secondary self-crosslinking network, respectively. Molecular weight and precursor concentration significantly affected physichemical properties and biological functions of the hydrogels. Although this HSMSSA gel (0.1 M Da, 10 mg/mL) had moderate injectability, preferable mechanical properties, and good proliferative ability of chondrocytes in vitro, and could greatly promote cartilaginous tissue formation in vivo, the lack of adhesion sites resulted in ineffective connections among newborn cell clusters. The biomimetic injectable di-self-crosslinking blend hydrogel improved chondrocyte adhesion and proliferation by combined injectable HSMSSA and bioactive Col I, especially for multiple ascending gene expression levels associated with hyaline cartilage formation and polyproteoglycan secretion.

Objective. To reveal the influence of functional mobility of connective-tissue structures on the development and course of intra-articular pathology of the knee joint. Materials and methods. Patients (n = 279) with intra-articular pathology of the knee joint after the arthroscopic treatment were examined. The state of functional mobility of connective-tissue structures was diagnosed using the express method for dysplastic state of connective tissues in conditions of ambulatory-polyclinic service. Statistical analysis was carried out with Pirson correlation criteria. Results. The paper is devoted to analysis of the influence of functional connective-tissue structure mobility on the development and course of intra-articular pathology of the knee joint. Subjects with low functional mobility of the connective-tissue structures were established to be more often subjected to chondromalacia of cartilaginous tissue of the femoral medial condyle and injury of the medial meniscus. Such pathology as slipping patella, Goff body hypertrophy and knee joint chondromatosis are typical for subjects with hypermobility of the connective-tissue structures. Conclusions. High degree of functional mobility of connective-tissue structures significantly contributes to preservation of cartilaginous cover of the femoral medial condyle, even with concomitant pathology of the knee joint available. For men with high functional mobility of connective-tissue structures, the most common diseases are knee joint chondromatosis and Goff body hypertrophy, and for women slipping patella. For women with low functional mobility of connective-tissue structures, the most typical is pathology of the medial meniscus.

Introduction Cutting the cost of manufacturing is important for extending the use of tissue-engineered therapeutic products. The present study aimed to develop a simple method for fabrication of cartilaginous tissues for regenerative therapy, utilizing the phenomenon where human articular chondrocytes grow thickness-wise and spontaneously form three-dimensionally thick tissues. Methods Normal human articular chondrocytes (NHACs) were cultured with varying concentrations of transforming growth factor beta 1 (TGF-β1) and/or fibroblast growth factor-2 (FGF-2) to optimize the culture condition for thickness-wise growth of chondrocytes. Next, the tissues grown in the optimal condition were subjected to re-differentiation culture in attached and detached states to assess differentiation capacity by evaluating secreted factors, histological analysis, and a gene expression assay. Results NHACs grew thickness-wise efficiently in the presence of 1 ng/mL TGF-β1 and 10 ng/mL FGF-2. After two weeks of culture, NHACs grew with 11-fold higher thickness and 16-fold higher cell number compared to cells which were neither treated with TGF-β1 nor with FGF-2. These thickness-wise-grown chondrocytes could be re-differentiated by a differentiation medium according to the increase in melanoma inhibitory activity (MIA) and positive safranin-O staining. Interestingly, the cartilaginous gene expression was considerably different between the attached and detached conditions even in the same culture medium, indicating the necessity of detachment and shrinkage to achieve further differentiation. Conclusions Spontaneous thickness-wise growth might provide a simple tissue-engineering method for manufacturing cartilaginous 3D tissues., Graphical abstract Image 1, Highlights • Chondrocytes grow thickness-wise efficiently in the presence of FGF-2 and TGF-β1 to medium. • Differentiation medium leads to MIA secretion and Safranin-O staining. • Detachment from culture surface promoted differentiation of chondrocytes.

The shortage of organ supply reduces the success rate of organ transplantation. Hence, tissue regeneration has been initiated with the intention of improving the available treatment modalities. Articular cartilage is a suitable tissue for this purpose due to its limited self-heal ability. This study aims to evaluate the cartilaginous properties of in vivo constructs formed using chondrocytes transfected with the combination of sry (sex determining region y)-box9 (SOX9) and telomerase reverse transcriptase (TERT) genes (SOX9/TERT-transfected chondrocytes) seeded on a three-dimensional (3D) poly(lactic-co-glycolic) acid (PLGA)-based scaffold. The rabbit’s articular chondrocytes (n=6) were transfected with SOX9 and TERT genes via lipofection. The non-transfected chondrocyte (NTC) was used as a control. A total of 1×105 cells were seeded on a PLGA and PLGA/fibrin hybrid scaffolds to form constructs. The resulted constructs were SOX9/TERT-PLGA/fibrin, NTC-PLGA/fibrin, SOX9/TERT-PLGA and, NTC-PLGA. All constructs were cultured for three weeks prior to subcutaneous implantation into the athymic mice for two and four weeks. The constructs’ structural and functional aspects were evaluated using macroscopic observation, compression-stress analysis, histology, quantitative sulphated glycosaminoglycan (sGAG) assay and cartilage-specific genes (ACAN, COL2A1, SOX9), TERT, and MMP13 expression analysis. The constructs demonstrated a cartilage-like appearance. The constructs’ rigidity corresponded to the homogenous cells and extracellular matrix distribution in the week-4 constructs. Correspondingly, the cartilaginous matrix components were visualised at the pericellular matrix region of the construct, supported by the increment of quantitative sGAG content. The SOX9/TERT-PLGA/fibrin exhibited better genes expression and cartilaginous phenotypes than the other construct groups. The SOX9/TERT-PLGA/fibrin construct facilitated cartilaginous tissue formation.

Humic acids are characterized by chelating properties due to which they are able to influence the mineral metabolism in animals. Rabbits have species-specific characteristics of mineral metabolism, which distinguishes them from most species of mammals. For the experiment, 16 rabbits of 45-day-old of Hyplus breed were divided into the experimental and control group of 8 rabbits in each. The animals were 15 days in the preparatory period. The rabbits of the experimental group received humic acid additive individually with water in the amount of 5 mg/kg, the rabbits of the control group received pure water in the same amount. The ability of humic acid additive to increase the content of calcium, ionized calcium, iron, to correct the content of copper and zinc, and also to increase the activity of alkaline phosphatase in the blood serum of rabbits of the experimental group has been proved. We observed the early effect of the humic acid additive on increasing the content of iron and calcium ionized and a later effect on the activity of alkaline phosphatase and an increase in the content of copper in the blood serum. The influence of humic acid additive on the distribution of calcium, phosphorus, manganese, copper and zinc in bone tissue is determined. According to a histological study, there was a significant increase in the number of layers of osteons and osteoblasts in the bone tissue of the femur, an increase in the number of osteons and osteoblasts in the bone tissue of the sternum and an increase in the number of columns of chondrocytes and an increase in the number of chondrocytes in the column in the cartilaginous tissue of the sternum. The results of a histological study of bone tissue, together with an increase in structural macroelements in it and redistribution of osteotropic trace elements in the blood and bone tissue suggest the possibility of using a humic acid additive to intensify the growth and mineralization of bone tissue, which can improve the state of bone tissue of rabbits in the postnatal period ontogenesis.

Background Although previous studies have suggested a stimulatory role of heparanase in physiological bone turnover, the potential therapeutic role of heparanase in bone healing has not been elucidated. The purpose of this study was to assess the effect of topical application of heparanase-1 on bone healing. Methods Two different dosages of recombinant mouse heparanase-1 and vehicle control were prepared and delivered via an osmotic pump to provide continuous topical infusion of the therapeutic reagent in a mouse bone defect model at the distal femoral metaphysis. The bone healing progress was evaluated by micro-computed tomography and histological examination at 7, 14, and 21 days after the bone defect was created. Results The peak of trabecular bone generation was achieved earlier than anticipated with the use of heparanase as measured by medullary bone volume fraction and trabecular number observed in micro-computed tomography, while the remodeling of trabecular bone to cortical bone was also achieved earlier than anticipated with the use of heparanase as measured by connectivity density. Histopathological observation revealed a higher frequency of the presence of cartilaginous tissue in the heparanase-treated groups. Both bone mineral density and cortical bone volume fraction showed the best healing outcome with low-dose heparanase, implying a biphasic effect of its mode of action. Conclusion These results indicated that with the appropriate dose of topical heparanase-1, the progress of bone healing could be accelerated in vivo.

Recently, many studies have focused on the repair and regeneration of damaged articular cartilage using tissue engineering. In tissue engineering therapy, cells are cultured in vitro to create a three-dimensional (3-D) tissue designed to replace the damaged cartilage. Although tissue engineering is a useful approach to regenerating cartilage, mechanical anisotropy has not been reconstructed from a cellular organization level. This study aims to create mechanically anisotropic cartilaginous tissue using dielectrophoretic cell patterning and gel-sheet lamination. Bovine chondrocytes were patterned in a hydrogel to form line-array cell clusters via negative dielectrophoresis (DEP). The results indicate that the embedded chondrocytes remained viable and reconstructed cartilaginous tissue along the patterned cell array. Moreover, the agarose gel, in which chondrocytes were patterned, demonstrated mechanical anisotropy. In summary, our DEP cell patterning and gel-sheet lamination techniques would be useful for reconstructing mechanically anisotropic cartilage tissues.

PURPOSE: The purpose of this study was to characterize the influence of SDF-1 on cell migration/adhesion and temporal gene expression of human cartilage mesenchymal progenitor cells (C-PCs); and to utilize SDF-1 conditioned mesenchymal progenitors to stimulate reintegration of human meniscus fibrocartilage breaks. MATERIALS AND METHODS: Characterization of SDF-1-induced cell migration was achieved using hydroxypropyl cellulose (HPC) scaffolds pretreated with SDF-1. Fluorescence microscopy and cell counting were used to visualize and quantify the extent of cell migration into scaffolds, respectively. Relative mRNA expression analysis was used to characterize the temporal effects of SDF-1 on C-PCs. Tissue reintegration experiments were conducted using cylindrical human meniscal tissue punches, which were then placed back together with an HPC scaffold embedded with C-PCs. Tensile testing was used to evaluate the extent of tissue reintegration stimulated by human mesenchymal progenitors. RESULTS: C-PCs migrate into scaffolds in response to SDF-1 with the same efficiency as mesenchymal progenitors from human marrow (BM-MSCs). SDF-1 treatment of C-PCs did not significantly alter the expression of early and late stage chondrogenic differentiation genes. Scaffolds containing SDF-1 preconditioned C-PCs successfully adhered to fibrocartilage breaks and migrated from the scaffold into the tissue. Tensile testing demonstrated that SDF-1 preconditioned C-PCs stimulate reintegration of fibrocartilage tears. CONCLUSION: C-PCs migrate in response to SDF-1. Exposure to SDF-1 does not significantly alter the unique mRNA profile of C-PCs that make them desirable for cartilaginous tissue repair applications. SDF-1 pretreated mesenchymal progenitors successfully disperse into injured tissues to help facilitate tissue reintegration.

Cartilage lacks basic repair mechanisms and thus surgical interventions are necessary to treat lesions. Minimally-invasive arthroscopic procedures require the development of injectable biomaterials to support chondrogenesis of implanted cells. However, most cartilage tissue engineering approaches rely on pre-culture of scaffolds in media containing growth factors (GFs) such as transforming growth factor (TGF)-β1, which are crucial for cartilage formation and homeostasis. GFs media-supplementation is incompatible with injectable approaches and has led to a knowledge gap about optimal dose of GFs and release profiles needed to achieve chondrogenesis. This study aims to determine the optimal loading and release kinetics of TGF-β1 bound to an engineered GAG hydrogel to promote optimal cartilaginous matrix production in absence of TGF-β1 media-supplementation. We show that heparin, a GAG known to bind a wide range of GFs, covalently conjugated to a hyaluronan hydrogel, leads to a sustained release of TGF-β1. Using this heparin-conjugated hyaluronan hydrogel, 0.25 to 50 ng TGF-β1 per scaffold was loaded and cell viability, proliferation and cartilaginous matrix deposition of the encapsulated chondroprogenitor cells were measured. Excellent chondrogenesis was found when 5 ng TGF-β1 per scaffold and higher were used. We also demonstrate the necessity of a sustained release of TGF-β1, as no matrix deposition is observed upon a burst release. In conclusion, our biomaterial loaded with an optimal initial dose of 5 ng/scaffold TGF-β1 is a promising injectable material for cartilage repair, with potentially increased safety due to the low, locally administered GF dose. Statement of Significance Cartilage cell-based products are dependent on exogenous growth factor supplementation in order for proper tissue maturation. However, for a one-step repair of defects without need for expensive tissue maturation, an injectable, growth factor loaded formulation is required. Here we show development of an injectable hyaluronan hydrogel, which achieves a sustained release of TGF-β1 due to covalent conjugation of heparin. These grafts matured into cartilaginous tissue in the absence of growth factor supplementation. Additionally, this system allowed us to screen TGF-β1 concentrations to determine the mimimum amount of growth factor required for chondrogenesis. This study represents a critical step towards development of a minimally-invasive, arthroscopic treatment for cartilage lesions.

Articular cartilage has limited capacity for regeneration and when damaged cannot be repaired with currently available metallic or synthetic implants. We aim to bioengineer a microfibre-reinforced hydrogel that can capture the zonal depth-dependent mechanical properties of native cartilage, and simultaneously support neo-cartilage formation. With this goal, a sophisticated bi-layered microfibre architecture, combining a densely distributed crossed fibre mat (superficial tangential zone, STZ) and a uniform box structure (middle and deep zone, MDZ), was successfully manufactured via melt electrospinning and combined with a gelatin–methacrylamide hydrogel. The inclusion of a thin STZ layer greatly increased the composite construct's peak modulus under both incongruent (3.2-fold) and congruent (2.1-fold) loading, as compared to hydrogels reinforced with only a uniform MDZ structure. Notably, the stress relaxation response of the bi-layered composite construct was comparable to the tested native cartilage tissue. Furthermore, similar production of sulphated glycosaminoglycans and collagen II was observed for the novel composite constructs cultured under mechanical conditioning w/o TGF-ß1 supplementation and in static conditions w/TGF-ß1 supplementation, which confirmed the capability of the novel composite construct to support neo-cartilage formation upon mechanical stimulation. To conclude, these results are an important step towards the design and manufacture of biomechanically competent implants for cartilage regeneration. Statement of Significance: Damage to articular cartilage results in severe pain and joint disfunction that cannot be treated with currently available implants. This study presents a sophisticated bioengineered bi-layered fibre reinforced cell-laden hydrogel that can approximate the functional mechanical properties of native cartilage. For the first time, the importance of incorporating a viable superficial tangential zone (STZ) – like structure to improve the load-bearing properties of bioengineered constructs, particularly when in-congruent surfaces are compressed, is demonstrated. The present work also provides new insights for the development of implants that are able to promote and guide new cartilaginous tissue formation upon physiologically relevant mechanical stimulation.

In Ukraine due to an increasing harmful effect of environmental factors on the human organism, especially in the period of the intrauterine development, the problem has become an important issue. Investigation of the dynamics of changes in the topography of structures of organs and complexes of organs during the prenatal period of human ontogenesis with the aim to determine the interrelations and interaction of the shape-generating processes on the spatial-temporal orientation of the anatomical structures as well as finding the time and morphological preconditions of possible occurrence of variants in their structure and congenital defects is one of the important scientific directions in anatomical field. 10 specimens of the nasal area of 7-8week pre-fetuses, regularities of their development, formation and morphological transformations of the human nasal septum were examined by means of micro- and morphometry. During the pre-fetal period the rudiment of the nasal septum was found to be presented by the mesenchyme covered externally with the high columnar epithelium with the nuclei of a spherical or oval shape. The mesenchyme cells are located more densely in the central part of the nasal septum rudiment forming an aggregation. The anterior and median thirds of the nasal septum are merged with the primary palate, and its posterior part hangs down into the primary oral cavity. In pre-fetuses at the end of the 7th week of development (19,5-20,0 mm long) the subepithelial blood network is presented by the vascular columns of a capillary type 8,0±0,5 mcm in diameter. During the 8th week of the intrauterine life a layer of the subcartilaginous tissue appears in the central part of the nasal septum due to differentiation of the mesenchyme cells. The mesenchyme cells are concentrated directly near the lower part of the posterior third of the nasal septum looking like a pair formation in the form of laminae – vomer rudiment. At the end of the 8th week of the intrauterine development the subcartilaginous tissue of the nasal septum is transformed into the immature cartilaginous tissue. The rudiment of the cuneopalatine artery (24,0±0,5 mcm in diameter) is also determined in the posterior portion of the lateral wall of the primary nasal cavity.

Introduction This study was performed with the aim of detecting the interaction between cartilaginous and bone tissues in the process of mandible morphogenesis in human embryos. Material and Methods Whole-mount skeletal preparations, stained with Alcian blue and Alizarin red, as well as serial histological sections impregnated with silver and stained with hematoxylin and eosin were studied. A total of 48 human embryos/fetuses from 5 to 29 weeks of age were used. Results In the mandible anlage, in addition to the woven bone, cartilaginous and chondroid tissues are present. Cartilaginous tissue islets are localized at the tip of the condylar and coronoid processes and also in the region of the mandibular symphysis. The chondroid tissue is incorporated into the bone in the region of the mandibular symphysis, along the edge of the coronoid process, and also in the alveolar part of the mandible. Conclusion Meckel’s cartilage participates in the formation of the mandibular body, and its remains persist in the area of the mandibular symphysis until the second half of the prenatal development.

Objective Although tissue engineering is a promising option for articular cartilage repair, it has been challenging to generate functional cartilaginous tissue. While the synthetic response of chondrocytes can be influenced by various means, most approaches treat chondrocytes as a homogeneous population that would respond similarly. However, isolated cells heterogeneously progress through the cell cycle, which can affect macromolecular biosynthesis. As it is possible to synchronize cells within discrete cell cycle phases, the purpose of this study was to investigate the effects of cell cycle synchronization on the chondrogenic potential of primary articular chondrocytes. Design Different methods of cell synchronization (serum starvation, thymidine, nocodazole, aphidicolin, and RO-3306) were tested for their ability to synchronize primary articular chondrocytes during the process of cell isolation. Cells (unsynchronized and synchronized) were then encapsulated in alginate gels, cultured for 4 weeks, and analyzed for their structural and biochemical properties. Results The double-thymidine method yielded the highest level of cell purity, with cells synchronized in S phase. While the cells started to lose synchronization after 24 hours, tissue constructs developed from initially S phase synchronized cells had significantly higher glycosaminoglycan and collagen II amounts than those developed using unsynchronized cells. Conclusions Initial synchronization led to long-term changes in cartilaginous tissue formation. This effect was postulated to be due to the rapid auto-induction of TGF-βs by actively dividing S phase cells, thereby stimulating chondrogenesis. Cell synchronization methods may also be applied in conjunction with redifferentiation methods to improve the chondrogenic potential of dedifferentiated or diseased chondrocytes.

Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs, and dense connective tissues (i.e., ligaments and tendons) is essential for the maintenance of spinal alignment. We show that the adhesion G protein-coupled receptor ADGRG6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine alignment in mice. Furthermore, we show that ADGRG6 regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with a cAMP agonist could mirror aspects of receptor function in culture, thus defining core pathways for regulating these axial cartilaginous and connective tissues. As ADGRG6 is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis., eLife, 10, ISSN:2050-084X

Acellular amniotic membrane (AM) has been studied, with promising results on the reconstruction of lesioned tissues, and has become an attractive approach for tracheal repair. This study aimed to evaluate the repair of the trachea with human umbilical cord mesenchymal stem cells (hucMSCs) differentiated in chondrocytes, grown on an experimental model. Tracheal defects were induced by surgical tracheostomy in 30 New Zealand rabbits, and the acellular amniotic membrane, with or without cells, was covering the defect. The hucMSCs were isolated and cultivated with chondrogenic differentiation over the culture of 14 days, and then grown on the AM. In this study, the AM was biocompatible and hucMSCs differentiated into chondrocytes. Our results demonstrated an important role for AM with cultured cells in the promotion of immature collagen, known to produce tissue regeneration. In addition, cartilaginous tissue was found at the tracheal defects, demonstrated by immunohistology results. This study suggests that this biomaterial implantation can be an effective future therapeutic alternative for patients with tracheal injury.

Osteochondroma usually arises from the metaphyseal region of growing bones. The occurrence of extraskeletal osteochondroma is rare with very few case reports. Para-articular osteochondroma is a type of extraskeletal osteochondroma. It frequently occurs around the knee, usually at infrapatellar Hoffa’s fat pad. It is usually intracapsular but extrasynovial and arises from the capsule and connective tissues due to osteocartilaginous metaplasia. We present a case of 19-years male with anterior knee pain for 3 years, swelling, and deformity of the knee with flexion limitation for one year. Radiography revealed ovoid, corticated lesion free from adjoining bones. Mass interpreted as benign, so planned for excision. Well circumscribed nodule excised from the medial parapatellar approach. Histology revealed cartilaginous tissues surrounded by fibrous tissues with scattered enchondral ossification.Postoperatively and subsequent follow-up resulted in pain-free joint, complete recovery of range of motion with no clinicoradiological evidence of recurrence.

Tissue engineered constructs have the potential to respond to the unmet medical need of treating deep osteochondral defects. However, current tissue engineering strategies struggle in the attempt to create patterned constructs with biologically distinct functionality. In this work, a developmentally-inspired modular approach is proposed, whereby distinct cartilaginous organoids are used as living building blocks. First, a hierarchical construct was created, composed of three layers of cartilaginous tissue intermediates derived from human periosteum-derived cells: (i) early (SOX9), (ii) mature (COL2) and (iii) (pre)hypertrophic (IHH, COLX) phenotype. Subcutaneous implantation in nude mice generated a hybrid tissue containing one mineralized and one non-mineralized part. However, the non-mineralized part was represented by a collagen type I positive fibrocartilage-like tissue. To engineer a more stable articular cartilage part, iPSC-derived cartilage microtissues (SOX9, COL2; IHH neg) were generated. Subcutaneous implantation of assembled iPSC-derived cartilage microtissues resulted in a homogenous cartilaginous tissue positive for collagen type II but negative for osteocalcin. Finally, iPSC-derived cartilage microtissues in combination with the pre-hypertrophic cartilage organoids (IHH, COLX) could form dual tissues consisting of i) a cartilaginous safranin O positive and ii) a bony osteocalcin positive region upon subcutaneous implantation, corresponding to the pre-engineered zonal pattern. The assembly of functional building blocks, as presented in this work, opens possibilities for the production of complex tissue engineered implants by embedding zone-specific functionality through the use of pre-programmed living building blocks. ispartof: BIOMATERIALS vol:273 ispartof: location:Netherlands status: published

Cellular communication network factor (CCN) 3 is one of the classical members of the CCN family, which are characterized by common molecular structures and multiple functionalities. Although this protein was discovered as a gene product overexpressed in a truncated form in nephroblastoma, recent studies have revealed its physiological roles in the development and homeostasis of mammalian species, in addition to its pathological association with a number of diseases. Cartilage is a tissue that creates most of the bony parts and cartilaginous tissues that constitute the human skeleton, in which CCN3 is also differentially produced to exert its molecular missions therein. In this review article, after the summary of the molecular structure and function of CCN3, recent findings on the regulation of ccn3 expression and the roles of CCN3 in endochondral ossification, cartilage development, maintenance and disorders are introduced with an emphasis on the metabolic regulation and function of this matricellular multifunctional molecule.

SummaryAdolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs and dense connective tissues (e.g., ligaments and tendons), are essential for maintenance of spinal alignment. We show that the G-coupled protein receptorAdgrg6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine morphology. We show thatAdgrg6regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with an cAMP agonist was able to mirror aspects of receptor function in culture defining core pathways for regulation of these axial connective tissues. AsADGRG6is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis.HighlightsKnockout mice lackingAdgrg6function in the tendons and ligaments of the spine develop perinatal-onset thoracic scoliosis.Loss ofAdgrg6function in cartilaginous tissues of the discs contribute to the incidence and severity of scoliosis.The loss ofAdgrg6function in spine tissues provide a model of construct validity for human adolescent idiopathic scoliosisFine tuning of the biomechanical properties of dense connective tissues is essential for maintaining spine alignment.

Temporomandibular disorders (TMD) are diseases involving the temporomandibular joint (TMJ), masticatory muscles, and osseous components. TMD has a high prevalence, with an estimated 4.8% of the U.S. population experiencing signs and symptoms, and represents a financial burden to both individuals and society. During TMD progression, the most frequently affected site is the condylar cartilage. Comprising both fibrous and cartilaginous tissues, condylar cartilage has restricted cell numbers but lacks a vascular supply and has limited regenerative properties. In 2016, a novel stem cell niche containing a reservoir of fibrocartilage stem cells (FCSCs) was discovered in the condylar cartilage of rats. Subsequently, FCSCs were identified in mouse, rabbit, and human condylar cartilage. Unlike mesenchymal stem cells or other tissue-specific stem/progenitor cells, FCSCs play a unique role in the development and regeneration of fibrocartilage. More importantly, engraftment treatment of FCSCs has been successfully applied in animal models of TMD. In this context, FCSCs play a major role in the regeneration of newly formed cartilage. Furthermore, FCSCs participate in the regeneration of intramembranous bone by interacting with endothelial cells in bone defects. This evidence highlights the potential of FCSCs as an ideal stem cell source for the regeneration of oral maxillofacial tissue. This review is intended to detail the current knowledge of the characteristics and function of FCSCs in the TMJ, as well as the potential therapeutic applications of FCSCs. A deep understanding of the properties of FCSCs can thus inform the development of promising, biologically based strategies for TMD in the future.

Van Neck-Odelberg disease (VND) is a rare benign pediatric skeletal abnormality characterized by hyperostosis of the ischiopubic synchondrosis (IPS) and clinical symptoms.1,2,3 IPS is a strip of cartilaginous tissue between the superomedial pubic and posterolateral ischial ossification centers, which develops into the inferior pubic rami and ischial tuberosity, respectively.

Despite the potential of tissue engineering approaches for cartilage repair, a major shortcoming is the low biosynthetic response of chondrocytes. While different strategies have been investigated to upregulate tissue formation, a novel approach may be to control nutrient metabolism. Although known for their anaerobic metabolism of glucose, chondrocytes are more synthetically active when cultured under conditions that elicit mixed aerobic-anaerobic metabolism. Here, we postulate this metabolic switch induces hypoxia inducible factor 1α (HIF-1α) signaling leading to improved tissue growth. Transition to different metabolic states can result in the pooling of intracellular metabolites, several of which can stabilize HIF-1α by interfering with proline-hydroxylase-2 (PHD2). Chondrocytes cultured under increased media availability accelerated tissue deposition (2.2 to 3.5-fold) with the greatest effect occurring at intermediate volumes (2 mL/106 cells). Under higher media volumes, metabolism switched from anaerobic to mixed aerobic-anaerobic. At and beyond this transition, maximal changes in PHD2 activity (- 45%), HIF-1α protein expression (8-fold increase), and HIF-1 gene target expression were observed (2.0 to 2.7-fold increase). Loss-of-function studies using YC-1 (to degrade HIF-1α) confirmed the involvement of HIF-1 signaling under these conditions. Lastly, targeted metabolomic studies of glucose metabolites (14 in total) revealed that both intracellular lactate and succinate correlated with PHD2 activity. Although both metabolites can inhibit PHD2, this effect can most likely be attributed to lactate as succinate was only present in trace amounts. However, addition work (e.g., 13C flux analyses) are required to confirm this assertion. Nevertheless, by harnessing this newly identified metabolic switch, functional engineered cartilage implants may be developed without the need for sophisticated methods which would allow for improved translation into the clinical realm.

Federal University of Par?. Institute of Biological Sciences. Bel?m, PA, Brazil. Federal University of Par?. Institute of Biological Sciences. Bel?m, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Microscopia. Ananindeua, PA, Brasil. Federal University of Par?. Department of Mechanical Engineering. Bel?m, PA, Brazil. Federal University of Par?. Institute of Biological Sciences. Bel?m, PA, Brazil. Federal University of Par?. Department of Nutrition. Bel?m, PA, Brazil. Campinas State University. National Institute of Science and Technology in Biofabrication. Chemical Engineering Department. Campinas, SP, Brazil. Federal University of Par?. Institute of Biological Sciences. Bel?m, PA, Brazil / Campinas State University. National Institute of Science and Technology in Biofabrication. Chemical Engineering Department. Campinas, SP, Brazil. Despite the many advances in the field of regenerative medicine, the use of biomaterials to repair the cartilaginous tissue's traumatic injuries is still a challenge. Based on that, this research focuses on the semi-interpenetrating polymeric scaffolds (SIPNs) based PLLA and PHEMA. The material was produced in the solvent absence as a strategy to outline the critical limitations of hydrophilicity and the lack of biological interaction of biomaterials with the surrounding tissues. To this end, the thermal properties and chemical structure were characterized using thermogravimetric analysis and spectroscopy in the Fourier transform infrared. Besides, we assessed porosity and tissue interactions by scanning electron microscopy, and the influence of this in the swelling capacity tests. In vitro and in vivo assays and histological analysis were developed to assess the biocompatibility of SIPNs scaffolds. The results showed good thermal stability, porosity in the range of 53.73 to 94.34??m, absence of toxicity, and excellent in vitro cell adhesion and growth. The biomaterial also promoted a great template for cell migration and spreading in vivo, with high cartilaginous tissue remodeling. SIPNs scaffolds demonstrated its biocompatibility and potential use in tissue engineering.

Background : Anophthalmia is a rare, congenital condition, defined as the complete absence of the eye bulb due to inadequate growth of the vesicle or optic dome. The malformation can be primary (in the absence of complete), secondary (in the presence of only residual tissue), or degenerative (in which the eye begins to form, but for some reason, it begins to degenerate). This condition is rare in dogs, cats, cattle, and sheep. Microscopic evaluation of orbital tissue for identification is always recommended. The aim of this study was to report a case of bilateral anophthalmia in a domestic cat. Case : A feline male, healthy, Maine Coon breed with 60 days of life was attended at the one veterinary private clinic. The cat, negative for FIV and FeLV, was born in a commercial cattery, belonging to his mother's third litter, healthy litter with the exception of this feline. He arrived with a complaint of not opening his eyelids, like the rest of the litter. In the clinical examination, it was found the normality of vital signs, absence of other visible anatomical abnormalities, only the ocular region was observed with closed eyelids. The initial suspicions were anophthalmia and microphthalmia. The patient was referred for an ocular ultrasound, which showed the complete absence of the right and left eye bulbs. The right and left orbital cavities had only a volume of soft, amorphous, and predominantly homogeneous tissue. After the ultrasound report, the patient underwent a surgical procedure to remove a fragment of tissue from the eye socket, which was sent for histopathological examination to confirm anophthalmia and discard the differential diagnosis of microphthalmia. Microscopy revealed immature, epithelial, and glandular tissue in the middle of discrete and moderate connective tissue, loosely arranged. In some fragments, cartilaginous tissue was also revealed. Thus, the histological findings are compatible with immature, pseudoformed tissues and without neoplastic characteristics. The diagnosis of secondary anophthalmia was reached with use of ultrasound and histological reports. Discussion : Congenital malformations in domestic cats are less frequent than in dogs, some of which are rare, and little reported. Secondary anophthalmia in the reported patient was confirmed by histological and ultrasound examination. Bilateral secondary anophthalmia is characterized by the absence of the eyeball, but with the presence of adjacent tissue. The animal was submitted to an ocular ultrasound examination and the complete absence of ocular bulbs was found. The differential diagnosis of microphthalmia was ruled out because there was no evidence of the eyeball. Microphthalmia is a common congenital ophthalmic disorder in veterinary medicine. Representative fragments were submitted to histopathological examination, where immature, epithelial tissue was found. In some fragments sent for analysis, cartilaginous tissue was observed. The histological findings are compatible with immature, pseudoformed tissues, thus verifying bilateral congenital anophthalmia in the reported animal. The clinical examination in these cases serves to ensure that the animal does not have any other congenital changes, allowing a favorable prognosis in puppies. Based on the information presented, the animal in this study has bilateral secondary congenital anophthalmia, with a favorable prognosis for the patient to live with certain normality, with quality and well-being.

Articular cartilage consists of hyaline cartilage, is a major constituent of the human musculoskeletal system and has critical functions in frictionless joint movement and articular homoeostasis. Osteoarthritis (OA) is an inflammatory disease of articular cartilage, which promotes joint degeneration. Although it affects millions of people, there are no satisfying therapies that address this disease at the molecular level. Therefore, tissue regeneration approaches aim at modifying chondrocyte biology to mitigate the consequences of OA. This requires appropriate biochemical and biophysical stimulation of cells. Regarding the latter, mechanotransduction of chondrocytes and their precursor cells has become increasingly important over the last few decades. Mechanotransduction is the transformation of external biophysical stimuli into intracellular biochemical signals, involving sensor molecules at the cell surface and intracellular signalling molecules, so-called mechano-sensors and -transducers. These signalling events determine cell behaviour. Mechanotransducing ion channels and gap junctions additionally govern chondrocyte physiology. It is of great scientific and medical interest to induce a specific cell behaviour by controlling these mechanotransduction pathways and to translate this knowledge into regenerative clinical therapies. This review therefore focuses on the mechanotransduction properties of integrins, cadherins and ion channels in cartilaginous tissues to provide perspectives for cartilage regeneration.

With the worldwide increase of fisheries, fish wastes have had a similar increase, alternatively they can be seen as a source of novel substances for the improvement of society&rsquo, s wellbeing. Elasmobranchs are a subclass fished in high amounts, with some species being mainly bycatch. They possess an endoskeleton composed mainly by cartilage, from which chondroitin sulfate is currently obtained. Their use as a viable source for extraction of type II collagen has been hypothesized with the envisaging of a biomedical application, namely in biomaterials production. In the present work, raw cartilage from shark (Prionace glauca) and ray (Zeachara chilensis and Bathyraja brachyurops) was obtained from a fish processing company and submitted to acidic and enzymatic extractions, to produce acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). From all the extractions, P. glauca PSC had the highest yield (3.5%), followed by ray ASC (0.92%), ray PSC (0.50%), and P. glauca ASC (0.15%). All the extracts showed similar properties, with the SDS-PAGE profiles being compatible with the presence of both type I and type II collagens. Moreover, the collagen extracts exhibited the competence to maintain their conformation at human basal temperature, presenting a denaturation temperature higher than 37 &deg, C. Hydrogels were produced using P. glauca PSC combined with shark chondroitin sulfate, with the objective of mimicking the human cartilage extracellular matrix. These hydrogels were cohesive and structurally-stable at 37 &deg, C, with rheological measurements exhibiting a conformation of an elastic solid when submitted to shear strain with a frequency up to 4 Hz. This work revealed a sustainable strategy for the valorization of fisheries&rsquo, by-products, within the concept of a circular economy, consisting of the use of P. glauca, Z. chilensis, and B. brachyurops cartilage for the extraction of collagen, which would be further employed in the development of hydrogels as a proof of concept of its biotechnological potential, ultimately envisaging its use in marine biomaterials to regenerate damaged cartilaginous tissues.

The term choristoma describes the growth of normal tissue in an abnormal location (Barnes et al., 2005) [1] Cartilaginous Choristoma is the proliferation of cartilaginous tissue in Choristoma. Here we report a two-year-old girl child who presented with hard swelling on the tongue. The child was investigated, and complete excision was done. The excised specimen was sent for histopathology, which reported as cartilaginous Choristoma of the tongue.