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This study has reviewed the many roles of lumican as a biomarker of tissue pathology in health and disease. Lumican is a structure regulatory proteoglycan of collagen-rich tissues, with cell instructive properties through interactions with a number of cell surface receptors in tissue repair, thereby regulating cell proliferation, differentiation, inflammation and the innate and humoral immune systems to combat infection. The exponential increase in publications in the last decade dealing with lumican testify to its role as a pleiotropic biomarker regulatory protein. Recent findings show lumican has novel roles as a biomarker of the hypercoagulative state that occurs in SARS CoV-2 infections; thus, it may also prove useful in the delineation of the complex tissue changes that characterize COVID-19 disease. Lumican may be useful as a prognostic and diagnostic biomarker of long COVID disease and its sequelae.

Abstract Animal models have been invaluable in the identification of molecular events occurring in and contributing to intervertebral disc (IVD) degeneration and important therapeutic targets have been identified. Some outstanding animal models (murine, ovine, chondrodystrophoid canine) have been identified with their own strengths and weaknesses. The llama/alpaca, horse and kangaroo have emerged as new large species for IVD studies, and only time will tell if they will surpass the utility of existing models. The complexity of IVD degeneration poses difficulties in the selection of the most appropriate molecular target of many potential candidates, to focus on in the formulation of strategies to effect disc repair and regeneration. It may well be that many therapeutic objectives should be targeted simultaneously to effect a favorable outcome in human IVD degeneration. Use of animal models in isolation will not allow resolution of this complex issue and a paradigm shift and adoption of new methodologies is required to provide the next step forward in the determination of an effective repairative strategy for the IVD. AI has improved the accuracy and assessment of spinal imaging supporting clinical diagnostics and research efforts to better understand IVD degeneration and its treatment. Implementation of AI in the evaluation of histology data has improved the usefulness of a popular murine IVD model and could also be used in an ovine histopathological grading scheme that has been used to quantify degenerative IVD changes and stem cell mediated regeneration. These models are also attractive candidates for the evaluation of novel anti‐oxidant compounds that counter inflammatory conditions in degenerate IVDs and promote IVD regeneration. Some of these compounds also have pain‐relieving properties. AI has facilitated development of facial recognition pain assessment in animal IVD models offering the possibility of correlating the potential pain alleviating properties of some of these compounds with IVD regeneration.

Abstract The purpose of this review was to evaluate data generated by animal models of intervertebral disc (IVD) degeneration published in the last decade and show how this has made invaluable contributions to the identification of molecular events occurring in and contributing to pain generation. IVD degeneration and associated spinal pain is a complex multifactorial process, its complexity poses difficulties in the selection of the most appropriate therapeutic target to focus on of many potential candidates in the formulation of strategies to alleviate pain perception and to effect disc repair and regeneration and the prevention of associated neuropathic and nociceptive pain. Nerve ingrowth and increased numbers of nociceptors and mechanoreceptors in the degenerate IVD are mechanically stimulated in the biomechanically incompetent abnormally loaded degenerate IVD leading to increased generation of low back pain. Maintenance of a healthy IVD is, thus, an important preventative measure that warrants further investigation to preclude the generation of low back pain. Recent studies with growth and differentiation factor 6 in IVD puncture and multi‐level IVD degeneration models and a rat xenograft radiculopathy pain model have shown it has considerable potential in the prevention of further deterioration in degenerate IVDs, has regenerative properties that promote recovery of normal IVD architectural functional organization and inhibits the generation of inflammatory mediators that lead to disc degeneration and the generation of low back pain. Human clinical trials are warranted and eagerly anticipated with this compound to assess its efficacy in the treatment of IVD degeneration and the prevention of the generation of low back pain.

This review examines the roles of HS–proteoglycans (HS–PGs) in general, and, in particular, perlecan and syndecan as representative examples and their interactive ligands, which regulate physiological processes and cellular behavior in health and disease. HS–PGs are essential for the functional properties of tissues both in development and in the extracellular matrix (ECM) remodeling that occurs in response to trauma or disease. HS–PGs interact with a biodiverse range of chemokines, chemokine receptors, protease inhibitors, and growth factors in immune regulation, inflammation, ECM stabilization, and tissue protection. Some cell regulatory proteoglycan receptors are dually modified hybrid HS/CS proteoglycans (betaglycan, CD47). Neurexins provide synaptic stabilization, plasticity, and specificity of interaction, promoting neurotransduction, neurogenesis, and differentiation. Ternary complexes of glypican-1 and Robbo–Slit neuroregulatory proteins direct axonogenesis and neural network formation. Specific neurexin–neuroligin complexes stabilize synaptic interactions and neural activity. Disruption in these interactions leads to neurological deficits in disorders of functional cognitive decline. Interactions with HS–PGs also promote or inhibit tumor development. Thus, HS–PGs have complex and diverse regulatory roles in the physiological processes that regulate cellular behavior and the functional properties of normal and pathological tissues. Specialized HS–PGs, such as the neurexins, pikachurin, and Eyes-shut, provide synaptic stabilization and specificity of neural transduction and also stabilize the axenome primary cilium of phototoreceptors and ribbon synapse interactions with bipolar neurons of retinal neural networks, which are essential in ocular vision. Pikachurin and Eyes–Shut interactions with an α-dystroglycan stabilize the photoreceptor synapse. Novel regulatory roles for HS–PGs controlling cell behavior and tissue function are expected to continue to be uncovered in this fascinating class of proteoglycan.

This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles in repair biology. Perlecan is ubiquitous, occurring in vascular, cartilaginous, adipose, lymphoreticular, bone and bone marrow stroma and in neural tissues. Perlecan has roles in angiogenesis, tissue development and extracellular matrix stabilization in mature weight bearing and tensional tissues. Perlecan contributes to mechanosensory properties in cartilage through pericellular interactions with fibrillin-1, type IV, V, VI and XI collagen and elastin. Perlecan domain I - FGF, PDGF, VEGF and BMP interactions promote embryonic cellular proliferation, differentiation, and tissue development. Perlecan domain II, an LDLR-like domain interacts with lipids, Wnt and Hedgehog morphogens. Perlecan domain III binds FGF-7 and 18 and has roles in the secretion of perlecan. Perlecan domain IV, an immunoglobulin repeat domain, has cell attachment and matrix stabilizing properties. Perlecan domain V promotes tissue repair through interactions with VEGF, VEGF-R2 and α2β1 integrin. Perlecan domain-V LG1-LG2 and LG3 fragments antagonize these interactions. Perlecan domain V promotes reconstitution of the blood brain barrier damaged by ischemic stroke and is neurogenic and neuroprotective. Perlecan-VEGF-VEGFR2, perlecan-FGF-2 and perlecan-PDGF interactions promote angiogenesis and wound healing. Perlecan domain I, III and V interactions with platelet factor-4 and megakaryocyte and platelet inhibitory receptor promote adhesion of cells to implants and scaffolds in vascular repair. Perlecan localizes acetylcholinesterase in the neuromuscular junction and is of functional significance in neuromuscular control. Perlecan mutation leads to Schwartz-Jampel Syndrome, functional impairment of the biomechanical properties of the intervertebral disc, variable levels of chondroplasia and myotonia. A greater understanding of the functional working of the neuromuscular junction may be insightful in therapeutic approaches in the treatment of neuromuscular disorders. Tissue engineering of salivary glands has been undertaken using bioactive peptides (TWSKV) derived from perlecan domain IV. Perlecan TWSKV peptide induces differentiation of salivary gland cells into self-assembling acini-like structures that express salivary gland biomarkers and secrete α-amylase. Perlecan also promotes chondroprogenitor stem cell maturation and development of pluripotent migratory stem cell lineages, which participate in diarthrodial joint formation, and early cartilage development. Recent studies have also shown that perlecan is prominently expressed during repair of adult human articular cartilage. Perlecan also has roles in endochondral ossification and bone development. Perlecan domain I hydrogels been used in tissue engineering to establish heparin binding growth factor gradients that promote cell migration and cartilage repair. Perlecan domain I collagen I fibril scaffolds have also been used as an FGF-2 delivery system for tissue repair. With the availability of recombinant perlecan domains, the development of other tissue repair strategies should emerge in the near future. Perlecan co-localization with vascular elastin in the intima, acts as a blood shear-flow endothelial sensor that regulates blood volume and pressure and has a similar role to perlecan in canalicular fluid, regulating bone development and remodeling. This complements perlecan’s roles in growth plate cartilage and in endochondral ossification to form the appendicular and axial skeleton. Perlecan is thus a ubiquitous, multifunctional, and pleomorphic molecule of considerable biological importance. A greater understanding of its diverse biological roles and functional repertoires during tissue development, growth and disease will yield valuable insights into how this impressive proteoglycan could be utilized successfully in repair biology.

Pentosan polysulfate (PPS), a small semi-synthetic highly sulfated heparan sulfate (HS)-like molecule, shares many of the interactive properties of HS. The aim of this review was to outline the potential of PPS as an interventional therapeutic protective agent in physiological processes affecting pathological tissues. PPS is a multifunctional molecule with diverse therapeutic actions against many disease processes. PPS has been used for decades in the treatment of interstitial cystitis and painful bowel disease, it has tissue-protective properties as a protease inhibitor in cartilage, tendon and IVD, and it has been used as a cell-directive component in bioscaffolds in tissue engineering applications. PPS regulates complement activation, coagulation, fibrinolysis and thrombocytopenia, and it promotes the synthesis of hyaluronan. Nerve growth factor production in osteocytes is inhibited by PPS, reducing bone pain in osteoarthritis and rheumatoid arthritis (OA/RA). PPS also removes fatty compounds from lipid-engorged subchondral blood vessels in OA/RA cartilage, reducing joint pain. PPS regulates cytokine and inflammatory mediator production and is also an anti-tumor agent that promotes the proliferation and differentiation of mesenchymal stem cells and the development of progenitor cell lineages that have proven to be useful in strategies designed to effect repair of the degenerate intervertebral disc (IVD) and OA cartilage. PPS stimulates proteoglycan synthesis by chondrocytes in the presence or absence of interleukin (IL)-1, and stimulates hyaluronan production by synoviocytes. PPS is thus a multifunctional tissue-protective molecule of potential therapeutic application for a diverse range of disease processes.

Flavonoids are a biodiverse family of dietary compounds that have antioxidant, anti-inflammatory, antiviral, and antibacterial cell protective profiles. They have received considerable attention as potential therapeutic agents in biomedicine and have been widely used in traditional complimentary medicine for generations. Such complimentary medical herbal formulations are extremely complex mixtures of many pharmacologically active compounds that provide a therapeutic outcome through a network pharmacological effects of considerable complexity. Methods are emerging to determine the active components used in complimentary medicine and their therapeutic targets and to decipher the complexities of how network pharmacology provides such therapeutic effects. The gut microbiome has important roles to play in the generation of bioactive flavonoid metabolites retaining or exceeding the antioxidative and anti-inflammatory properties of the intact flavonoid and, in some cases, new antitumor and antineurodegenerative bioactivities. Certain food items have been identified with high prebiotic profiles suggesting that neutraceutical supplementation may be beneficially employed to preserve a healthy population of bacterial symbiont species and minimize the establishment of harmful pathogenic organisms. Gut health is an important consideration effecting the overall health and wellbeing of linked organ systems. Bioconversion of dietary flavonoid components in the gut generates therapeutic metabolites that can also be transported by the vagus nerve and systemic circulation to brain cell populations to exert a beneficial effect. This is particularly important in a number of neurological disorders (autism, bipolar disorder, AD, PD) characterized by effects on moods, resulting in depression and anxiety, impaired motor function, and long-term cognitive decline. Native flavonoids have many beneficial properties in the alleviation of inflammation in tissues, however, concerns have been raised that therapeutic levels of flavonoids may not be achieved, thus allowing them to display optimal therapeutic effects. Dietary manipulation and vagal stimulation have both yielded beneficial responses in the treatment of autism spectrum disorders, depression, and anxiety, establishing the vagal nerve as a route of communication in the gut-brain axis with established roles in disease intervention. While a number of native flavonoids are beneficial in the treatment of neurological disorders and are known to penetrate the blood–brain barrier, microbiome-generated flavonoid metabolites (e.g., protocatechuic acid, urolithins, γ-valerolactones), which retain the antioxidant and anti-inflammatory potency of the native flavonoid in addition to bioactive properties that promote mitochondrial health and cerebrovascular microcapillary function, should also be considered as potential biotherapeutic agents. Studies are warranted to experimentally examine the efficacy of flavonoid metabolites directly, as they emerge as novel therapeutic options.

Heparan sulfate is a ubiquitous, variably sulfated interactive glycosaminoglycan that consists of repeating disaccharides of glucuronic acid and glucosamine that are subject to a number of modifications (acetylation, de-acetylation, epimerization, sulfation). Variable heparan sulfate chain lengths and sequences within the heparan sulfate chains provide structural diversity generating interactive oligosaccharide binding motifs with a diverse range of extracellular ligands and cellular receptors providing instructional cues over cellular behaviour and tissue homeostasis through the regulation of essential physiological processes in development, health, and disease. heparan sulfate and heparan sulfate-PGs are integral components of the specialized glycocalyx surrounding cells. Heparan sulfate is the most heterogeneous glycosaminoglycan, in terms of its sequence and biosynthetic modifications making it a difficult molecule to fully characterize, multiple ligands also make an elucidation of heparan sulfate functional properties complicated. Spatio-temporal presentation of heparan sulfate sulfate groups is an important functional determinant in tissue development and in cellular control of wound healing and extracellular remodelling in pathological tissues. The regulatory properties of heparan sulfate are mediated via interactions with chemokines, chemokine receptors, growth factors and morphogens in cell proliferation, differentiation, development, tissue remodelling, wound healing, immune regulation, inflammation, and tumour development. A greater understanding of these HS interactive processes will improve therapeutic procedures and prognoses. Advances in glycosaminoglycan synthesis and sequencing, computational analytical carbohydrate algorithms and advanced software for the evaluation of molecular docking of heparan sulfate with its molecular partners are now available. These advanced analytic techniques and artificial intelligence offer predictive capability in the elucidation of heparan sulfate conformational effects on heparan sulfate-ligand interactions significantly aiding heparan sulfate therapeutics development.

This study has reviewed the naturally occurring bioadhesives produced in marine and freshwater aqueous environments and in the mucinous exudates of some terrestrial animals which have remarkable properties providing adhesion under difficult environmental conditions. These bioadhesives have inspired the development of medical bioadhesives with impressive properties that provide an effective alternative to suturing surgical wounds improving closure and healing of wounds in technically demanding tissues such as the heart, lung and soft tissues like the brain and intestinal mucosa. The Gecko has developed a dry-adhesive system of exceptional performance and has inspired the development of new generation re-usable tapes applicable to many medical procedures. The silk of spider webs has been equally inspiring to structural engineers and materials scientists and has revealed innovative properties which have led to new generation technologies in photonics, phononics and micro-electronics in the development of wearable biosensors. Man made products designed to emulate the performance of these natural bioadhesive molecules are improving wound closure and healing of problematic lesions such as diabetic foot ulcers which are notoriously painful and have also found application in many other areas in biomedicine. Armed with information on the mechanistic properties of these impressive biomolecules major advances are expected in biomedicine, micro-electronics, photonics, materials science, artificial intelligence and robotics technology.

The aim of this review is to highlight the beneficial attributes of flavonoids, a diverse family of widely-distributed polyphenolic phytochemicals that have beneficial cell and tissue protective properties. Phytochemicals are widely distributed in plants, herbs and shrubs used in traditional complimentary medical formulations for centuries. The bioactive components that convey beneficial medicinal effects in these complex herbal preparations are now being identified using network pharmacology and molecular docking procedures that identify their molecular targets. Flavonoids have anti-oxidant, anti-inflammatory, antiviral, antibacterial and anti-cancer properties that have inspired the development of potent multifunctional derivatised flavonoids of improved efficacy. The antiviral properties of flavonoids and the emergence of the severe acute respiratory syndrome (SARS-CoV-2) pandemic has resulted in a resurgence of interest in phytochemicals in the search for efficacious compounds that can prevent viral infection or replication, with many promising plant compounds identified. Promising semi-synthetic flavonoid derivatives have also been developed that inhibit multiple pathological neurodegenerative processes; these offer considerable promise in the treatment of diseases of cognitive decline. Clinical trials are currently being undertaken to evaluate the efficacy of dietary supplements rich in flavonoids for the treatment of virally-mediated diseases. Such trials are expected to identify flavonoids with cell and tissue protective properties that can be harnessed in biomedical applications that may serve as supportive adjunctive procedures to conventional anti-viral drug therapies against diseases such as COVID-19.

Chondroitin sulfate (CS) is the most abundant and widely distributed glycosaminoglycan (GAG) in the human body. As a component of proteoglycans (PGs) it has numerous roles in matrix stabilization and cellular regulation. This chapter highlights the roles of CS and CS-PGs in the central and peripheral nervous systems (CNS/PNS). CS has specific cell regulatory roles that control tissue function and homeostasis. The CNS/PNS contains a diverse range of CS-PGs which direct the development of embryonic neural axonal networks, and the responses of neural cell populations in mature tissues to traumatic injury. Following brain trauma and spinal cord injury, a stabilizing CS-PG-rich scar tissue is laid down at the defect site to protect neural tissues, which are amongst the softest tissues of the human body. Unfortunately, the CS concentrated in gliotic scars also inhibits neural outgrowth and functional recovery. CS has well known inhibitory properties over neural behavior, and animal models of CNS/PNS injury have demonstrated that selective degradation of CS using chondroitinase improves neuronal functional recovery. CS-PGs are present diffusely in the CNS but also form denser regions of extracellular matrix termed perineuronal nets which surround neurons. Hyaluronan is immobilized in hyalectan CS-PG aggregates in these perineural structures, which provide neural protection, synapse, and neural plasticity, and have roles in memory and cognitive learning. Despite the generally inhibitory cues delivered by CS-A and CS-C, some CS-PGs containing highly charged CS disaccharides (CS-D, CS-E) or dermatan sulfate (DS) disaccharides that promote neural outgrowth and functional recovery. CS/DS thus has varied cell regulatory properties and structural ECM supportive roles in the CNS/PNS depending on the glycoform present and its location in tissue niches and specific cellular contexts. Studies on the fruit fly, Drosophila melanogaster and the nematode Caenorhabditis elegans have provided insightful information on neural interconnectivity and the role of the ECM and its PGs in neural development and in tissue morphogenesis in a whole organism environment.

This narrative review highlights the complexities of the gut microbiome and health-promoting properties of prebiotic xylans metabolized by the gut microbiome. In animal husbandry, prebiotic xylans aid in the maintenance of a healthy gut microbiome. This prevents the colonization of the gut by pathogenic organisms obviating the need for dietary antibiotic supplementation, a practice which has been used to maintain animal productivity but which has led to the emergence of antibiotic resistant bacteria that are passed up the food chain to humans. Seaweed xylan-based animal foodstuffs have been developed to eliminate ruminant green-house gas emissions by gut methanogens in ruminant animals, contributing to atmospheric pollution. Biotransformation of pentosan polysulfate by the gut microbiome converts this semi-synthetic sulfated disease-modifying anti-osteoarthritic heparinoid drug to a prebiotic metabolite that promotes gut health, further extending the therapeutic profile and utility of this therapeutic molecule. Xylans are prominent dietary cereal components of the human diet which travel through the gastrointestinal tract as non-digested dietary fibre since the human genome does not contain xylanolytic enzymes. The gut microbiota however digest xylans as a food source. Xylo-oligosaccharides generated in this digestive process have prebiotic health-promoting properties. Engineered commensal probiotic bacteria also have been developed which have been engineered to produce growth factors and other bioactive factors. A xylan protein induction system controls the secretion of these compounds by the commensal bacteria which can promote gut health or, if these prebiotic compounds are transported by the vagal nervous system, may also regulate the health of linked organ systems via the gut–brain, gut–lung and gut–stomach axes. Dietary xylans are thus emerging therapeutic compounds warranting further study in novel disease prevention protocols.

Mucin-like glycoproteins have established roles in epithelial boundary protection and lubricative roles in some tissues. This mini-review illustrates alternative functional roles which rely on keratan sulphate and sialic acid modifications to mucin glycopolymers which convey charge properties suggestive of novel electroconductive properties not previously ascribed to these polymers. Many tumour cells express mucin-like glycopolymers modified with highly sulphated keratan sulphate and sialic which can be detected using diagnostic biosensors. The mucin-like keratan sulphate glycopolymer present in the ampullae of lorenzini is a remarkable sensory polymer which elasmobranch fish (sharks, rays, skate) use to detect weak electrical fields emitted through muscular activity of prey fish. Information on the proton gradients is conveyed to neuromast cells located at the base of the ampullae and mechanotransduced to neural networks. This ampullae keratan sulphate sensory gel is the most sensitive proton gradient detection polymer known in nature. This process is known as electrolocation, and allows the visualization of prey fish under conditions of low visibility. The bony fish have similar electroreceptors located along their lateral lines which consist of neuromast cells containing sensory hairs located within a cupula which contains a sensory gel polymer which detects distortions in fluid flow in channels within the lateral lines and signals are sent back to neural networks providing information on the environment around these fish. One species of dolphin, the Guiana dolphin, has electrosensory pits in its bill with similar roles to the ampullae but which have evolved from its vibrissal system. Only two terrestrial animals can undertake electrolocation, these are the Duck-billed platypus and long and short nosed Echidna. In this case the electrosensor is a highly evolved innervated mucous gland. The platypus has 40,000 electroreceptors around its bill through which it electrolocates food species. The platypus has poor eyesight, is a nocturnal feeder and closes its eyes, nostrils and ears when it hunts, so electrolocation is an essential sensory skill. Mammals also have sensory cells containing stereocilia which are important in audition in the organ of corti of the cochlea and in olfaction in the olfactory epithelium. The rods and cones of the retina also have an internal connecting cilium with roles in the transport of phototransduced chemical signals and activation of neurotransmitter release to the optic nerve. Mucin-like glycopolymer gels surround the stereocilia of these sensory hair cells but these are relatively poorly characterized however they deserve detailed characterization since they may have important functional attributes.

The aim of this study was to illustrate recent developments in neural repair utilizing hyaluronan as a carrier of olfactory bulb stem cells and in new bioscaffolds to promote neural repair. Hyaluronan interacts with brain hyalectan proteoglycans in protective structures around neurons in perineuronal nets, which also have roles in the synaptic plasticity and development of neuronal cognitive properties. Specialist stem cell niches termed fractones located in the sub-ventricular and sub-granular regions of the dentate gyrus of the hippocampus migrate to the olfactory bulb, which acts as a reserve of neuroprogenitor cells in the adult brain. The extracellular matrix associated with the fractone stem cell niche contains hyaluronan, perlecan and laminin α5, which regulate the quiescent recycling of stem cells and also provide a means of escaping to undergo the proliferation and differentiation to a pluripotent migratory progenitor cell type that can participate in repair processes in neural tissues. Significant improvement in the repair of spinal cord injury and brain trauma has been reported using this approach. FGF-2 sequestered by perlecan in the neuroprogenitor niche environment aids in these processes. Therapeutic procedures have been developed using olfactory ensheathing stem cells and hyaluronan as a carrier to promote neural repair processes. Now that recombinant perlecan domain I and domain V are available, strategies may also be expected in the near future using these to further promote neural repair strategies.

The aim of this study was to highlight the roles of perlecan in the regulation of the development of the rudiment developmental cartilages and growth plate cartilages, and also to show how perlecan maintains permanent articular cartilage homeostasis. Cartilage rudiments are transient developmental templates containing chondroprogenitor cells that undergo proliferation, matrix deposition, and hypertrophic differentiation. Growth plate cartilage also undergoes similar changes leading to endochondral bone formation, whereas permanent cartilage is maintained as an articular structure and does not undergo maturational changes. Pericellular and extracellular perlecan-HS chains interact with growth factors, morphogens, structural matrix glycoproteins, proteases, and inhibitors to promote matrix stabilization and cellular proliferation, ECM remodelling, and tissue expansion. Perlecan has mechanotransductive roles in cartilage that modulate chondrocyte responses in weight-bearing environments. Nuclear perlecan may modulate chromatin structure and transcription factor access to DNA and gene regulation. Snail-1, a mesenchymal marker and transcription factor, signals through FGFR-3 to promote chondrogenesis and maintain Acan and type II collagen levels in articular cartilage, but prevents further tissue expansion. Pre-hypertrophic growth plate chondrocytes also express high Snail-1 levels, leading to cessation of Acan and CoI2A1 synthesis and appearance of type X collagen. Perlecan differentially regulates FGF-2 and FGF-18 to maintain articular cartilage homeostasis, rudiment and growth plate cartilage growth, and maturational changes including mineralization, contributing to skeletal growth.

Cancer metastasis is the dissemination of tumor cells to new sites, resulting in the formation of secondary tumors. This process is complex and is spatially and temporally regulated by intrinsic and extrinsic factors. One important extrinsic factor is the extracellular matrix, the non-cellular component of tissues. Heparan sulfate proteoglycans (HSPGs) are constituents of the extracellular matrix, and through their heparan sulfate chains and protein core, modulate multiple events that occur during the metastatic cascade. This review will provide an overview of the role of the extracellular matrix in the events that occur during cancer metastasis, primarily focusing on perlecan. Perlecan, a basement membrane HSPG is a key component of the vascular extracellular matrix and is commonly associated with events that occur during the metastatic cascade. Its contradictory role in these events will be discussed and we will highlight the recent advances in cancer therapies that target HSPGs and their modifying enzymes.

Background. The extracellular matrix of the PNS/CNS is unusual in that it is dominated by glycosaminoglycans, especially hyaluronan, whose space filling and hydrating properties make essential contributions to the functional properties of this tissue. Hyaluronan has a relatively simple structure but its space-filling properties ensure micro-compartments are maintained in the brain ultrastructure, ensuring ionic niches and gradients are maintained for optimal cellular function. Hyaluronan has cell-instructive, anti-inflammatory properties and forms macro-molecular aggregates with the lectican CS-proteoglycans, forming dense protective perineuronal net structures that provide neural and synaptic plasticity and support cognitive learning. Aims. To highlight the central nervous system/peripheral nervous system (CNS/PNS) and its diverse extracellular and cell-associated proteoglycans that have cell-instructive properties regulating neural repair processes and functional recovery through interactions with cell adhesive molecules, receptors and neuroregulatory proteins. Despite a general lack of stabilising fibrillar collagenous and elastic structures in the CNS/PNS, a sophisticated dynamic extracellular matrix is nevertheless important in tissue form and function. Conclusions. This review provides examples of the sophistication of the CNS/PNS extracellular matrix, showing how it maintains homeostasis and regulates neural repair and regeneration.

The recent discovery of nuclear and perinuclear perlecan in annulus fibrosus and nucleus pulposus cells and its known matrix stabilizing properties in tissues introduces the possibility that perlecan may also have intracellular stabilizing or regulatory roles through interactions with nuclear envelope or cytoskeletal proteins or roles in nucleosomal-chromatin organization that may regulate transcriptional factors and modulate gene expression. The nucleus is a mechano-sensor organelle, and sophisticated dynamic mechanoresponsive cytoskeletal and nuclear envelope components support and protect the nucleus, allowing it to perceive and respond to mechano-stimulation. This review speculates on the potential roles of perlecan in the nucleus based on what is already known about nuclear heparan sulphate proteoglycans. Perlecan is frequently found in the nuclei of tumour cells; however, its specific role in these diseased tissues is largely unknown. The aim of this review is to highlight probable roles for this intriguing interactive regulatory proteoglycan in the nucleus of normal and malignant cell types.

In this study, we review mechanoregulatory roles for perlecan in load-bearing connective tissues. Perlecan facilitates the co-acervation of tropoelastin and assembly of elastic microfibrils in translamellar cross-bridges which, together with fibrillin and elastin stabilise the extracellular matrix of the intervertebral disc annulus fibrosus. Pericellular perlecan interacts with collagen VI and XI to define and stabilize this matrix compartment which has a strategic position facilitating two-way cell-matrix communication between the cell and its wider extracellular matrix. Cues from the extracellular matrix are fed through this pericellular matrix back to the chondrocyte, allowing it to perceive and respond to subtle microenvironmental changes to regulate tissue homeostasis. Thus perlecan plays a key regulatory role in chondrocyte metabolism, and in chondrocyte differentiation. Perlecan acts as a transport proteoglycan carrying poorly soluble, lipid-modified proteins such as the Wnt or Hedgehog families facilitating the establishment of morphogen gradients that drive tissue morphogenesis. Cell surface perlecan on endothelial cells or osteocytes acts as a flow sensor in blood and the lacunar canalicular fluid providing feedback cues to smooth muscle cells regulating vascular tone and blood pressure, and the regulation of bone metabolism by osteocytes highlighting perlecan’s multifaceted roles in load-bearing connective tissues.

Articular cartilage is considered to have limited regenerative capacity, which has led to the search for therapies to limit or halt the progression of its destruction. Perlecan, a multifunctional heparan sulphate (HS) proteoglycan, promotes embryonic cartilage development and stabilises the mature tissue. We investigated the immunolocalisation of perlecan and collagen between donor-matched biopsies of human articular cartilage defects (n = 10 × 2) that were repaired either naturally or using autologous cell therapy, and with age-matched normal cartilage. We explored how the removal of HS from perlecan affects human chondrocytes in vitro. Immunohistochemistry showed both a pericellular and diffuse matrix staining pattern for perlecan in both natural and cell therapy repaired cartilage, which related to whether the morphology of the newly formed tissue was hyaline cartilage or fibrocartilage. Immunostaining for perlecan was significantly greater in both these repair tissues compared to normal age-matched controls. The immunolocalisation of collagens type III and VI was also dependent on tissue morphology. Heparanase treatment of chondrocytes in vitro resulted in significantly increased proliferation, while the expression of key chondrogenic surface and genetic markers was unaffected. Perlecan was more prominent in chondrocyte clusters than in individual cells after heparanase treatment. Heparanase treatment could be a means of increasing chondrocyte responsiveness to cartilage injury and perhaps to improve repair of defects.

This review examines aggrecan’s roles in developmental embryonic tissues, in tissues undergoing morphogenetic transition and in mature weight-bearing tissues. Aggrecan is a remarkably versatile and capable proteoglycan (PG) with diverse tissue context-dependent functional attributes beyond its established role as a weight-bearing PG. The aggrecan core protein provides a template which can be variably decorated with a number of glycosaminoglycan (GAG) side chains including keratan sulphate (KS), human natural killer trisaccharide (HNK-1) and chondroitin sulphate (CS). These convey unique tissue-specific functional properties in water imbibition, space-filling, matrix stabilisation or embryonic cellular regulation. Aggrecan also interacts with morphogens and growth factors directing tissue morphogenesis, remodelling and metaplasia. HNK-1 aggrecan glycoforms direct neural crest cell migration in embryonic development and is neuroprotective in perineuronal nets in the brain. The ability of the aggrecan core protein to assemble CS and KS chains at high density equips cartilage aggrecan with its well-known water-imbibing and weight-bearing properties. The importance of specific arrangements of GAG chains on aggrecan in all its forms is also a primary morphogenetic functional determinant providing aggrecan with unique tissue context dependent regulatory properties. The versatility displayed by aggrecan in biodiverse contexts is a function of its GAG side chains.

This study has identified keratan sulfate in fetal and adult rat spinal cord and vertebral connective tissues using the antibody BKS-1(+) which recognizes a reducing terminal N-acetyl glucosamine-6-sulfate neo-epitope exposed by keratanase-I digestion. Labeling patterns were correlated with those of lumican and keratocan using core protein antibodies to these small leucine rich proteoglycan species. BKS-1(+) was not immunolocalized in fetal spinal cord but was apparent in adult cord and was also prominently immunolocalized to the nucleus pulposus and inner annulus fibrosus of the intervertebral disc. Interestingly, BKS-1(+) was also strongly associated with vertebral body ossification centers of the fetal spine. Immunolocalization of lumican and keratocan was faint within the vertebral body rudiments of the fetus and did not correlate with the BKS-1(+) localization indicating that this reactivity was due to another KS-proteoglycan, possibly osteoadherin (osteomodulin) which has known roles in endochondral ossification. Western blotting of adult rat spinal cord and intervertebral discs to identify proteoglycan core protein species decorated with the BKS-1(+) motif confirmed the identity of 37 and 51 kDa BKS-1(+) positive core protein species. Lumican and keratocan contain low sulfation KS-I glycoforms which have neuroregulatory and matrix organizational properties through their growth factor and morphogen interactive profiles and ability to influence neural cell migration. Furthermore, KS has interactive capability with a diverse range of neuroregulatory proteins that promote neural proliferation and direct neural pathway development, illustrating key roles for keratocan and lumican in spinal cord development.

The aim of this study was to determine the role of the perlecan (Hspg2) heparan sulphate (HS) side chains on cell and matrix homeostasis in tail and Achilles tendons in 3 and 12 week old Hspg2 exon 3 null HS deficient (Hspg2Δ3 − ∕Δ3 −) and C57 BL/6 Wild Type (WT) mice. Perlecan has important cell regulatory and matrix organizational properties through HS mediated interactions with a range of growth factors and morphogens and with structural extracellular matrix glycoproteins which define tissue function and allow the resident cells to regulate tissue homeostasis. It was expected that ablation of the HS chains on perlecan would severely disrupt normal tendon organization and functional properties and it was envisaged that this study would better define the role of HS in normal tendon function and in tendon repair processes. Tail and Achilles tendons from each genotype were biomechanically tested (ultimate tensile stress (UTS), tensile modulus (TM)) and glycosaminoglycan (GAG) and collagen (hydroxyproline) compositional analyses were undertaken. Tenocytes were isolated from tail tendons from each mouse genotype and grown in monolayer culture. These cultures were undertaken in the presence of FGF-2 to assess the cell signaling properties of each genotype. Total RNA was isolated from 3–12 week old tail and Achilles tendons and qRT-PCR was undertaken to assess the expression of the following genes Vcan, Bgn, Dcn, Lum, Hspg2, Ltbp1, Ltbp2, Eln and Fbn1. Type VI collagen and perlecan were immunolocalised in tail tendon and collagen fibrils were imaged using transmission electron microscopy (TEM). FGF-2 stimulated tenocyte monolayers displayed elevated Adamts4, Mmp2, 3, 13 mRNA levels compared to WT mice. Non-stimulated tendon Col1A1, Vcan, Bgn, Dcn, Lum, Hspg2, Ltbp1, Ltbp2, Eln and Fbn1 mRNA levels showed no major differences between the two genotypes other than a decline with ageing while LTBP2 expression increased. Eln expression also declined to a greater extent in the perlecan exon 3 null mice (P

Heparan sulfate (HS) and heparin are glycosaminoglycans (GAGs) that are heterogeneous in nature, not only due to differing disaccharide combinations, but also their sulfate modifications. HS is well known for its interactions with various growth factors and cytokines; and heparin for its clinical use as an anticoagulant. Due to their potential use in tissue regeneration; and the recent adverse events due to contamination of heparin; there is an increased surge to produce these GAGs on a commercial scale. The production of HS from natural sources is limited so strategies are being explored to be biomimetically produced via chemical; chemoenzymatic synthesis methods and through the recombinant expression of proteoglycans. This review details the most recent advances in the field of HS/heparin synthesis for the production of low molecular weight heparin (LMWH) and as a tool further our understanding of the interactions that occur between GAGs and growth factors and cytokines involved in tissue development and repair.

This study reviewed aspects of the biology of two members of the glucosinolate family, namely sinigrin and glucoraphanin and their anti-tumour and antimicrobial properties. Sinigrin and glucoraphanin are converted by the β-sulphoglucosidase myrosinase or the gut microbiota into their bioactive forms, allyl isothiocyanate (AITC) and sulphoraphanin (SFN) which constitute part of a sophisticated defence system plants developed over several hundred million years of evolution to protect them from parasitic attack from aphids, ticks, bacteria or nematodes. Delivery of these components from consumption of cruciferous vegetables rich in the glucosinolates also delivers many other members of the glucosinolate family so the dietary AITCs and SFN do not act in isolation. In vitro experiments with purified AITC and SFN have demonstrated their therapeutic utility as antimicrobials against a range of clinically important bacteria and fungi. AITC and SFN are as potent as Vancomycin in the treatment of bacteria listed by the World Health Organisation as antibiotic-resistant “priority pathogens” and also act as anti-cancer agents through the induction of phase II antioxidant enzymes which inactivate potential carcinogens. Glucosinolates may be useful in the treatment of biofilms formed on medical implants and catheters by problematic pathogenic bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus and are potent antimicrobials against a range of clinically important bacteria and fungi. The glucosinolates have also been applied in the prevention of bacterial and fungal spoilage of food products in advanced atmospheric packaging technology which improves the shelf-life of these products.

The aim of this study was to review aspects of the pathobiology of the meniscus in health and disease and show how degeneration of the meniscus can contribute to deleterious changes in other knee joint components. The menisci, distinctive semilunar weight bearing fibrocartilages, provide knee joint stability, co-ordinating functional contributions from articular cartilage, ligaments/tendons, synovium, subchondral bone and infra-patellar fat pad during knee joint articulation. The meniscus contains metabolically active cell populations responsive to growth factors, chemokines and inflammatory cytokines such as interleukin-1 and tumour necrosis factor-alpha, resulting in the synthesis of matrix metalloproteases and A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS)-4 and 5 which can degrade structural glycoproteins and proteoglycans leading to function-limiting changes in meniscal and other knee joint tissues. Such degradative changes are hall-marks of osteoarthritis (OA). No drugs are currently approved that change the natural course of OA and translate to long-term, clinically relevant benefits. For any pharmaceutical therapeutic intervention in OA to be effective, disease modifying drugs will have to be developed which actively modulate the many different cell types present in the knee to provide a global therapeutic. Many individual and combinatorial approaches are being developed to treat or replace degenerate menisci using 3D printing, bioscaffolds and hydrogel delivery systems for therapeutic drugs, growth factors and replacement progenitor cell populations recognising the central role the menisci play in knee joint health.

Background: Cartilage regeneration requires a balance of anabolic and catabolic processes. Aim: To examine the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage, in cartilage development and in osteoarthritis (OA). Methods: Immunolocalization of FMOD and LUM in fetal foot and adult knee cartilages using an FMOD matrix metalloprotease (MMP)-13 neoepitope antibody (TsYG11) and C-terminal anti-FMOD (PR184) and anti-LUM (PR353) antibodies. The in vitro digestion of knee cartilage with MMP-13, A Disintegrin and Metalloprotease with Thrompospondin motifs (ADAMTS)-4 and ADAMTS-5, to assess whether FMOD and LUM fragments observed in Western blots of total knee replacement specimens could be generated. Normal ovine articular cartilage explants were cultured with interleukin (IL)-1 and Oncostatin-M (OSM) ± PGE3162689, a broad spectrum MMP inhibitor, to assess FMOD, LUM and collagen degradation. Results and Discussion: FMOD and LUM were immunolocalized in metatarsal and phalangeal fetal rudiment cartilages and growth plates. Antibody TsYG11 localized MMP-13-cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plates. FMOD was more prominently localized in the superficial cartilage of normal and fibrillated zones in OA cartilage. TsYG11-positive FMOD was located deep in the cartilage samples. Ab TsYG11 identified FMOD fragmentation in Western blots of normal and fibrillated cartilage extracts and total knee replacement cartilage. The C-terminal anti-FMOD, Ab PR-184, failed to identify FMOD fragmentation due to C-terminal processing. The C-terminal LUM, Ab PR-353, identified three LUM fragments in OA cartilages. In vitro digestion of human knee cartilage with MMP-13, ADAMTS-4 and ADAMTS-5 generated FMOD fragments of 54, 45 and 32 kDa similar to in blots of OA cartilage; LUM was less susceptible to fragmentation. Ab PR-353 detected N-terminally processed LUM fragments of 39, 38 and 22 kDa in 65⁻80-year-old OA knee replacement cartilage. FMOD and LUM were differentially processed in MMP-13, ADAMTS-4 and ADAMTS-5 digestions. FMOD was susceptible to degradation by MMP-13, ADAMTS-4 and to a lesser extent by ADAMTS-5; however, LUM was not. MMP-13-cleaved FMOD in metatarsal and phalangeal fetal rudiment and growth plate cartilages suggested roles in skeletogenesis and OA pathogenesis. Explant cultures of ovine cartilage stimulated with IL-1/OSM ± PGE3162689 displayed GAG loss on day 5 due to ADAMTS activity. However, by day 12, the activation of proMMPs occurred as well as the degradation of FMOD and collagen. These changes were inhibited by PGE3162689, partly explaining the FMOD fragments seen in OA and the potential therapeutic utility of PGE3162689.

Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. Discussion/Conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.