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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.

Disc degeneration and associated back and neck pain elicits a substantial burden on healthcare systems and the individuals affected, necessitating the development of novel therapeutic strategies. This goal can only be achieved by a better understanding of intervertebral disc development, homeostasis and pathogenesis. A number of genetic and in-bred murine models are reviewed to underscore the importance of the mouse as an animal model of choice for the assessment of intervertebral disc pathobiology. Appraisals of the differences between mouse and human musculoskeletal systems and proteoglycan structures are also included. A number of important target pathways and molecules have been identified, many of which are worthy of further examination, requiring that the activity of these be confirmed in large animal models and assessed in the context of therapeutic intervention.

Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved over several hundred million years of vertebrate evolution. IαI is a complex containing the proteoglycan bikunin to which heavy chain proteins are covalently attached to the chondroitin sulfate chain. Besides its matrix protective activity through protease inhibitory action, IαI family members interact with extracellular matrix molecules and most notably hyaluronan, inhibit complement, and provide cell regulatory functions. Recent evidence for the diverse roles of the IαI family in both biology and pathology is reviewed and gives insight into their pivotal roles in tissue homeostasis. In addition, the clinical uses of these molecules are explored, such as in the treatment of inflammatory conditions including sepsis and Kawasaki disease, which has recently been associated with severe acute respiratory syndrome coronavirus 2 infection in children

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.

Purpose Proprioceptive deficits are common in low back pain. The multifidus muscle undergoes substantial structural change after back injury, but whether muscle spindles are affected is unclear. This study investigated whether muscle spindles of the multifidus muscle are changed by intervertebral disc (IVD) degeneration in a large animal model. Methods IVD degeneration was induced by partial thickness annulus fibrosus lesion to the L3-4 IVD in nine sheep. Multifidus muscle tissue at L4 was harvested at six months after lesion, and from six age-/sex-matched naïve control animals. Muscle spindles were identified in Van Gieson’s-stained sections by morphology. The number, location and cross-sectional area (CSA) of spindles, the number, type and CSA of intrafusal fibers, and thickness of the spindle capsule were measured. Immunofluorescence assays examined Collagen I and III expression. Results Multifidus muscle spindles were located centrally in the muscle and generally near connective tissue. There were no differences in the number or location of muscle spindles after IVD degeneration and only changes in the CSA of nuclear chain fibers. The thickness of connective tissue surrounding the muscle spindle was increased as was the expression of Collagen I and III. Conclusion Changes to the connective tissue and collagen expression of the muscle spindle capsule are likely to impact their mechanical properties. Changes in capsule stiffness may impact the transmission of length change to muscle spindles and thus transduction of sensory information. This change in muscle spindle structure may explain some of the proprioceptive deficits identified with low back pain.

Compared to the other classes of glycosaminoglycans (GAGs), that is, chondroitin/dermatan sulfate, heparin/heparan sulfate and hyaluronan, keratan sulfate (KS), have the least known of its interactive properties. In the human body, the cornea and the brain are the two most abundant tissue sources of KS. Embryonic KS is synthesized as a linear poly-N-acetyllactosamine chain of d-galactose-GlcNAc repeat disaccharides which become progressively sulfated with development, sulfation of GlcNAc is more predominant than galactose. KS contains multi-sulfated high-charge density, monosulfated and non-sulfated poly-N-acetyllactosamine regions and thus is a heterogeneous molecule in terms of chain length and charge distribution. A recent proteomics study on corneal KS demonstrated its interactivity with members of the Slit-Robbo and Ephrin-Ephrin receptor families and proteins which regulate Rho GTPase signaling and actin polymerization/depolymerization in neural development and differentiation. KS decorates a number of peripheral nervous system/CNS proteoglycan (PG) core proteins. The astrocyte KS-PG abakan defines functional margins of the brain and is up-regulated following trauma. The chondroitin sulfate/KS PG aggrecan forms perineuronal nets which are dynamic neuroprotective structures with anti-oxidant properties and roles in neural differentiation, development and synaptic plasticity. Brain phosphacan a chondroitin sulfate, KS, HNK-1 PG have roles in neural development and repair. The intracellular microtubule and synaptic vesicle KS-PGs MAP1B and SV2 have roles in metabolite transport, storage, and export of neurotransmitters and cytoskeletal assembly. MAP1B has binding sites for tubulin and actin through which it promotes cytoskeletal development in growth cones and is highly expressed during neurite extension. The interactive capability of KS with neuroregulatory ligands indicate varied roles for KS-PGs in development and regenerative neural processes.

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.

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. View Full-Text\udKeywords: aggrecan; tissue morphogenesis; HNK-1 trisaccharide; glycosaminoglycan; cellular regulation; extracellular matrix

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.

Perlecan, or heparan sulfate proteoglycan 2 (HSPG2), is a ubiquitous heparan sulfate proteoglycan that has major roles in tissue and organ development and wound healing by orchestrating the binding and signaling of mitogens and morphogens to cells in a temporal and dynamic fashion. In this review, its roles in fibrosis are reviewed by drawing upon evidence from tissue and organ systems that undergo fibrosis as a result of an uncontrolled response to either inflammation or traumatic cellular injury leading to an over production of a collagen-rich extracellular matrix. This review focuses on examples of fibrosis that occurs in lung, liver, kidney, skin, kidney, neural tissues and blood vessels and its link to the expression of perlecan in that particular organ system.

The meniscal cartilages in the knee function to improve congruity of the medial and lateral femoro-tibial joints and play critical roles in load distribution and joint stability. Meniscal tears of various configurations are one of the most common conditions of the knee and are associated with an increased risk of developing osteoarthritis (OA). While this risk has been largely attributed to loss of the biomechanical functions of the menisci, there is accumulating evidence suggesting that other aspects of meniscal biology may play a role in determining the long-term consequences of meniscal damage for joint health. In this narrative review, we examine the existing literature and present some new data implicating synthesis and secretion of enzymes and other pro-catabolic mediators by injured and degenerate menisci, contributing to the pathological change in other knee joint tissues in OA.

Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-&alpha, 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 &alpha, 1-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 &alpha, 1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of &alpha, 1-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.

The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis.

Study design Longitudinal case-controlled animal study. Objective To investigate putative cellular mechanisms to explain structural changes in muscle and adipose and connective tissues of the back muscles after intervertebral disc (IVD) injury. Summary of background data Structural back muscle changes are ubiquitous with back pain/injury and considered relevant for outcome, but their exact nature, time course, and cellular mechanisms remain elusive. We used an animal model that produces phenotypic back muscle changes after IVD injury to study these issues at the cellular/molecular level. Methods Multifidus muscle was harvested from both sides of the spine at L1-L2 and L3-L4 IVDs in 27 castrated male sheep at 3 (n = 10) or 6 (n = 17) months after a surgical anterolateral IVD injury at both levels. Ten control sheep underwent no surgery (3 mo, n = 4; 6 mo, n = 6). Tissue was harvested at L4 for histological analysis of cross-sectional area of muscle and adipose and connective tissue (whole muscle), plus immunohistochemistry to identify proportion and cross-sectional area of individual muscle fiber types in the deepest fascicle. Quantitative polymerase chain reaction measured gene expression of typical cytokines/signaling molecules at L2. Results Contrary to predictions, there was no multifidus muscle atrophy (whole muscle or individual fiber). There was increased adipose and connective tissue (fibrotic proliferation) cross-sectional area and slow-to-fast muscle fiber transition at 6 but not 3 months. Within the multifidus muscle, increases in the expression of several cytokines (tumor necrosis factor α and interleukin-1β) and molecules that signal trophic/atrophic processes for the 3 tissue types (e.g., growth factor pathway [IGF-1, PI3k, Akt1, mTOR], potent tissue modifiers [calcineurin, PCG-1α, and myostatin]) were present. Conclusion This study provides cellular evidence that refutes the presence of multifidus muscle atrophy accompanying IVD degeneration at this intermediate time point. Instead, adipose/connective tissue increased in parallel with the expression of the genes that provide putative mechanisms for multifidus structural remodeling. This provides novel targets for pharmacological and physical interventions. Level of evidence N/A.

The intervertebral disc (IVD) is a major weight bearing structure that undergoes degenerative changes with aging limiting its ability to dissipate axial spinal loading in an efficient manner resulting in the generation of low back pain. Low back pain is a number one global musculoskeletal disorder with massive socioeconomic impact. The WHO has nominated development of mesenchymal stem cells and bioscaffolds to promote IVD repair as primary research objectives. There is a clear imperative for the development of strategies to effectively treat IVD defects. Early preclinical studies with mesenchymal stem cells in canine and ovine models have yielded impressive results in IVD repair. Combinatorial therapeutic approaches encompassing biomaterial and cell-based therapies promise significant breakthroughs in IVD repair in the near future.

We have developed an ovine meniscal explant model where the focal degradative events leading to characteristic fragmentation patterns of biglycan in human OA of the knee and hip, and evident in animal models of knee OA and IVD degeneration are reproduced in culture. Lateral and medial menisci were dissected into outer, mid and inner zones and established in explant culture±IL-1 (10ng/ml). The biglycan species present in conditioned media samples and in GuHCl extracts of tissues were examined by Western blotting using two C-terminal antibodies PR-85 and EF-Bgn. Clear differences were evident in the biglycan species in each meniscal tissue zone with the medial outer meniscus having lower biglycan levels and major fragments of 20, 28, 33 and 36, 39kDa. Similar fragmentation was detected in articular cartilage samples, 42-45kDa core protein species were also detected. Biglycan fragmentation was not as extensive in the IL-1 stimulated meniscal cultures with 36, 39, 42 and 45kDa biglycan species evident. Thus the medial meniscus outer zone displayed the highest levels of biglycan processing in this model and correlated with a major zone of meniscal remodelling in OA in man. Significantly, enzymatic digests of meniscal tissues with MMP-13, ADAMTS-4 and ADAMTS-5 have also generated similar biglycan species in-vitro. Zymography confirmed that the medial outer zone was the region of maximal MMP activity. This model represents a convenient system to recapitulate matrix remodelling events driven by IL-1 in pathological cartilages and in animal models of joint degeneration.

Heparan sulfate (HS) regulates diverse cell signalling events in intervertebral disc development and homeostasis. The aim of the present study was to investigate the effect of ablation of perlecan HS/CS on murine intervertebral disc development. Genetic models carrying mutations in genes encoding HS biosynthetic enzymes have identified multiple roles for HS in tissue homeostasis. In the present study, we utilised an

Purpose Intervertebral disk (IVD) lesion and its subsequent degeneration have a profound effect on the multifidus muscle. The subacute/early chronic phase of multifidus remodeling after IVD lesion has been proposed to be regulated by inflammatory processes. The balance between pro-inflammatory (M1) and anti-inflammatory (M2) macrophages plays an important role in maintaining tissue integrity after injury. The localization, polarization of macrophage subtypes and their mediation of the pro-inflammatory cytokine tumor necrosis factor (TNF) are unknown in paraspinal muscles during IVD degeneration. A sheep model of IVD degeneration was used to investigate the role of macrophages and TNF in the structural alterations that occur within the multifidus muscle. Methods Anterolateral lesions were induced at L3–4 IVD in sheep. Multifidus muscle tissue at L4 was harvested 3 and 6 months after lesion and used for immunofluorescence assays to examine total macrophage number, macrophage polarization between M1 and M2, and to assess the localization of TNF expression in muscle, adipose and connective tissues from injured and naïve control animals. Results A greater proportion of M1 macrophages is present in muscle at both 3 and 6 months after IVD lesion, and adipose tissue at 6 months. Total number of macrophages is unchanged. At 6 months, expression of TNF is increased in adipose and connective tissue and the proportion of TNF expressed by M1 macrophages is increased. Conclusions These data support the proposal that macrophages and TNF (pro-inflammatory cytokine) play an active role in the subacute/early chronic phase of remodeling in muscle, adipose and connective tissues of the multifidus during IVD degeneration. This presents a novel target for treatment.

The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin sulfate/dermatan sulfate (CS/DS) with selected functional molecules in neural tissues. Cell surface glycans and GAGs have evolved over millions of years to become cellular mediators which regulate fundamental aspects of cellular survival. The glycocalyx, which surrounds all cells, actuates responses to growth factors, cytokines and morphogens at the cellular boundary, silencing or activating downstream signaling pathways and gene expression. In this review, we have focused on interactions mediated by l-fucose, KS and CS/DS in the central and peripheral nervous systems. Fucose makes critical contributions in the area of molecular recognition and information transfer in the blood group substances, cytotoxic immunoglobulins, cell fate-mediated Notch-1 interactions, regulation of selectin-mediated neutrophil extravasation in innate immunity and CD-34-mediated new blood vessel development, and the targeting of neuroprogenitor cells to damaged neural tissue. Fucosylated glycoproteins regulate delivery of synaptic neurotransmitters and neural function. Neural KS proteoglycans (PGs) were examined in terms of cellular regulation and their interactive properties with neuroregulatory molecules. The paradoxical properties of CS/DS isomers decorating matrix and transmembrane PGs and the positive and negative regulatory cues they provide to neurons are also discussed.

From an evolutionary perspective keratan sulfate (KS) is the newest glycosaminoglycan (GAG) but the least understood. KS is a sophisticated molecule with a diverse structure, and unique functional roles continue to be uncovered for this GAG. The cornea is the richest tissue source of KS in the human body but the central and peripheral nervous systems also contain significant levels of KS and a diverse range of KS-proteoglycans with essential functional roles. KS also displays important cell regulatory properties in epithelial and mesenchymal tissues and in bone and in tumor development of diagnostic and prognostic utility. Corneal KS-I displays variable degrees of sulfation along the KS chain ranging from non-sulfated polylactosamine, mono-sulfated and disulfated disaccharide regions. Skeletal KS-II is almost completely sulfated consisting of disulfated disaccharides interrupted by occasional mono-sulfated N-acetyllactosamine residues. KS-III also contains highly sulfated KS disaccharides but differs from KS-I and KS-II through 2-O-mannose linkage to serine or threonine core protein residues on proteoglycans such as phosphacan and abakan in brain tissue. Historically, the major emphasis on the biology of KS has focused on its sulfated regions for good reason. The sulfation motifs on KS convey important molecular recognition information and direct cell behavior through a number of interactive proteins. Emerging evidence also suggest functional roles for the poly-N-acetyllactosamine regions of KS requiring further investigation. Thus further research is warranted to better understand the complexities of KS.

The development of bioscaffolds that incorporate chondroitin sulphate (CS) and their applications with progenitor and stem cells in cartilage, bone, cornea, skin, and neural repair are reviewed. CS is a heterogeneous structure due to the organisation of multiple CS disaccharide sulphation motifs, giving rise to a vast range of CS chain structures, and hence the wide range of biological activity. The incorporation of this biological molecule represents a significant advance in bioscaffold design and performance in tissue repair strategies. The intrinsic stem-cell directive properties of CS are covered in the context of tissue development, and the differing CS disaccharide motifs, referred to as the 'glyco-code'. These structural motifs contribute to stem cell proliferation and differentiation in the scaffold environment and improve outcomes in terms of tissue repair or regeneration worthy of future research.

Key events that occur during inflammation include the recruitment, adhesion, and transmigration of leukocytes from the circulation to the site of inflammation. These events are modulated by chemokines, integrins, and selectins and the interaction of these molecules with glycosaminoglycans, predominantly heparan sulfate (HS). The development of HS/heparin mimetics that interfere or inhibit the interactions that occur between glycosaminoglycans and modulators of inflammation holds great potential for use as anti-inflammatory therapeutics. This review will detail the role of HS in the events that occur during inflammation, their interaction and modulation of inflammatory mediators, and the current advances in the development of HS/heparin mimetics as anti-inflammatory biotherapeutics.

The aim of this study was to examine the complexity of the stem cell populations in the intervertebral disc (IVD) and understand their role in disc degeneration, with a view of determining whether the resident stem cells could be developed for therapeutic purposes to combat IVD degeneration. Stem cells have been isolated from disc and paradiscal tissues, including the notochord, annulus fibrosus (AF), nucleus pulposus (NP), cartilaginous endplate (CEP), ligamentum flavum, and vertebral body. Resident AF and NP cells are relatively sparsely distributed occurring as single or occasional doublet cells surrounded by an extensive extracellular matrix (ECM). Small clusters of 4-12 cells also occur close to annular lesions in experimental ovine and canine disc degeneration, these are indicative of an attempted repair response by resident stem cells. The rat IVD also has notochordal and peripheral cell populations in the outer AF, which express CS sulfation motifs (7-D-4, 4-C-3, 3-B-3[-]) characteristic of activated stem cells, the murine IVD also has a cell population in the outer AF adjacent to the vertebral growth plate with characteristics of a progenitor cell population. These have also been observed in rabbit, minipig, ovine, and human IVDs. Chondroid cell nests in the ovine NP may represent a progenitor/stem cell reserve. Such human chondroid cells express CS sulfation motifs (7-D-4, 4-C-3, 3-B-3[-]), cytokeratin-8 and 19, and CD cell surface markers typical of stem cells, including OCT3/4, CD105, CD90, STRO-1, NOTCH1, and JAGGED1. Similar stem cell populations are present in grade IV degenerate human IVDs. A greater understanding of the biology of this chondroid cell population may identify them as a therapeutic resource. A resident therapeutic cell type adapted to the demanding IVD environment may be advantageous in repair strategies.

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.

The present study was designed to ascertain how altered biomechanics in adolescent idiopathic scoliotic (AIS) intervertebral discs (IVDs) affected tissue compositions and aggrecan processing compared to age matched and aged human IVDs. Newborn, 2- and 10-year-old ovine IVDs were also examined. Aggrecan populations were separated by Sepharose CL2B chromatography, composite agarose polyacrylamide gel electrophoresis (CAPAGE) and identified by immunoblotting. The KS and CS content of IVD tissue extracts from AIS IVDs were compared with age-matched normal adolescent IVDs and with old human IVDs. Extracts from newborn, 2- and 10-year-old ovine IVDs were also examined in a similar manner. Adolescent idiopathic scoliotic IVD Aggrecan populations shared similar levels of polydispersity and aggregatability with hyaluronan as old IVD proteoglycans. CAPAGE demonstrated three aggrecan populations in AIS, aged human and ovine IVDs increased polydispersity and mobility in CAPAGE. AIS IVDs had GAG compositions similar to aged human and ovine IVDs. Sulphated KS (5-D-4) and chondroitin-6-sulphate, 3-B-3(+) were markers of tissue maturation, and chondroitin-4-sulphate, 2-B-6(+) was prominent in immature IVDs but its levels were lower in mature IVDs. Sulphated KS and 3-B-3(+) CS were prominently associated with IVD maturation and AIS IVDs, while the 2-B-6(+) CS isomer was associated with immature IVD tissues. The polydispersity of aggrecan in AIS IVDs, which was similar to in old human and ovine IVDs, reflected altered processing in the AIS IVDs in response to the biomechanical microenvironments the disc cells were exposed to in AIS IVDs. These slides can be retrieved under Electronic Supplementary Material.

The purpose of this study was to develop a quantitative histopathological scoring scheme to evaluate disc degeneration and regeneration using an ovine annular lesion model of experimental disc degeneration. Toluidine blue and Haematoxylin and Eosin (H&E) staining were used to evaluate cellular morphology: (i) disc structure/lesion morphology; (ii) proteoglycan depletion; (iii) cellular morphology; (iv) blood vessel in-growth; (v) cell influx into lesion; and (vi) cystic degeneration/chondroid metaplasia. Three study groups were examined: 5 × 5 mm lesion; 6 × 20 mm lesion; and 6 × 20 mm lesion plus mesenchymal stem cell (MSC) treatment. Lumbar intervertebral discs (IVDs) were scored under categories (i–vi) to provide a cumulative score, which underwent statistical analysis using STATA software. Focal proteoglycan depletion was associated with 5 × 5 mm annular rim lesions, bifurcations, annular delamellation, concentric and radial annular tears and an early influx of blood vessels and cells around remodeling lesions but the inner lesion did not heal. Similar features in 6 × 20 mm lesions occurred over a 3–6-month post operative period. MSCs induced a strong recovery in discal pathology with a reduction in cumulative histopathology degeneracy score from 15.2 to 2.7 (p = 0.001) over a three-month recovery period but no recovery in carrier injected discs.

Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 μg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 μg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 μg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P

Perlecan is a widely distributed, heparan sulphate proteoglycan with roles in the sequestration of FGFs, PDGF, VEGF through which it promotes cell proliferation and matrix production. Perlecan also stabilises extracellular matrices through interaction with a diverse range of matrix components. This study examined the distribution of perlecan in an ovine partial transection tendinopathy model. In normal tendon, perlecan was immunolocalised to small blood vessels in intrafascicular regions in the tendon-bone and muscle-tendon attachments and to linear arrays of oval shaped tenocytes in the tendon mid-region. Partial transection in the mid-tendon region significantly increased perlecan accumulation within the fascicles, in granulation tissue filling the transection site and in the tendon-bone and tendon-muscle attachments. The accumulation of perlecan in the transected tendon and its known roles in matrix stabilisation and cell proliferation indicate possible roles in tendon remodelling and repair. Perlecan domain-1 has been used as a growth factor delivery vehicle for FGF-2, BMP-2 and BMP-7 in regenerative medicine but has yet to be evaluated in infraspinatus tendon repair. A better understanding of perlecan's contributions to pathobiological processes in remodelling tendon may be useful in such regenerative strategies in the future.

The aim of this study was to immunolocalise type VI collagen and perlecan and determine their interactive properties in the intervertebral disc (IVD). Confocal laser scanning microscopy co-localised perlecan with type VI collagen as pericellular components of IVD cells and translamellar cross-bridges in ovine and murine IVDs. These cross-bridges were significantly less abundant in the heparin sulphate deficient Hspg2 exon 3 null mouse IVD than in wild type. This association of type VI collagen with elastic components provides clues as to its roles in conveying elastic recoil properties to annular tissues. Perlecan and type VI collagen were highly interactive in plasmon resonance studies. Pericellular colocalisation of perlecan and type VI collagen provides matrix stabilisation and cell-matrix communication which allows IVD cells to perceive and respond to perturbations in their biomechanical microenvironment. Perlecan, at the cell surface, provides an adhesive interface between the cell and its surrounding extracellular matrix. Elastic microfibrillar structures regulate tensional connective tissue development and function. The 2010 Global Burden of Disease study examined 291 disorders and identified disc degeneration and associated low back pain as the leading global musculoskeletal disorder emphasising its massive socioeconomic impact and the need for more effective treatment strategies. A greater understanding of how the IVD achieves its unique biomechanical functional properties is of great importance in the development of such therapeutic measures.

Study Design: Longitudinal case-control animal model. Objective: To investigate effects of mesenchymal stem cell (MSC) treatment on multifidus muscle remodeling after intervertebral disc (IVD) lesion. Summary of Background Data: Lesion and degeneration of IVDs cause structural remodeling of the multifidus muscle. Proinflammatory cytokines are thought to contribute. MSC treatment restores IVD health after lesion but its effects on surrounding tissues remains unknown. Using an animal model of IVD degeneration, we assessed the effects of MSC treatment of IVDs on the structural remodeling and cytokine expression within the multifidus muscle. Methods: An anterolateral lesion was performed on the L1–2, L3–4, and L5–6 IVDs in sheep. At either 4 (early treatment) or 12 (late treatment) weeks after IVD lesion, MSCs were injected into the lesioned IVD. Multifidus muscle was harvested from L2 (gene expression analysis) and L4 (histological analysis) at 3 or 6 months after IVD lesion and naïve controls for histological analysis of muscle, adipose, and connective tissue cross-sectional areas, and immunohistochemistry to study muscle fiber types. Real-time polymerase chain reactions quantified expression of tumor necrosis factor, interleukin-1β, and transforming growth factor-β1. Results: MSC treatment of IVD lesion prevented the increased adipose and connective tissue cross-sectional area expected after IVD lesion. MSC treatment did not prevent slow-to-fast muscle fiber type transformation. Gene expression of proinflammatory cytokines within the muscle was altered by the MSC treatment of IVD. Increased interleukin-1β expression was prevented in the early treatment group and tumor necrosis factor and transforming growth factor-β1 expression was upregulated at 6 months. Conclusion: Results show that although MSC treatment prevents fatty infiltration and fibrosis of the multifidus muscle after IVD lesion, it cannot prevent a muscle inflammatory response and muscle fiber transformation. These findings highlight the potential role of MSC therapy after IVD injury, but reveals that other interventions may also be necessary to optimize recovery of muscle. Level of Evidence: 4

This was an observational study where we examined the role of perlecan HS on the deposition of TGF-β1 in C57BL/6 and Hspg2(∆3-/∆3-) perlecan exon 3 null mouse skin. Despite its obvious importance in skin repair and tissue homeostasis no definitive studies have immunolocalised TGF-β1 in skin in WT or Hspg2(∆3-/∆3-) perlecan exon 3 null mice. Vertical parasagittal murine dorsal skin from 3, 6 and 12 week old C57BL/6 and Hspg2(∆3-/∆3-) mice were fixed in neutral buffered formalin, paraffin embedded and 4 μm sections stained with Mayers haematoxylin and eosin (HE). TGF-β1 was immunolocalised using a rabbit polyclonal antibody, heat retrieval and the Envision NovaRED detection system. Immunolocalisation of TGF-β1 differed markedly in C57BL/6 and Hspg2(∆3-/∆3-) mouse skin, ablation of exon 3 of Hspg2 resulted in a very severe reduction in the deposition of TGF-β1 in skin 3-12 weeks postnatally. The reduced deposition of TGF-β1 observed in the present study would be expected to impact detrimentally on the remodelling and healing capacity of skin in mutant mice compounding on the poor wound-healing properties already reported for perlecan exon 3 null mice due to an inability to signal with FGF-2 and promote angiogenic repair processes. TGF-β1 also has cell mediated effects in tissue homeostasis and matrix stabilisation a reduction in TGF-β1 deposition would therefore be expected to detrimentally impact on skin homeostasis in the perlecan mutant mice.

The role of hyaluronan (HA) oligosaccharides in disc cell-mediated matrix metalloproteinase (MMP) and anabolic gene expression in vitro and annular repair in vivo were examined. Monolayer and alginate bead cultures of ovine intervertebral disc cells were stimulated with 10-12 mer hyaluronan oligosaccharides (HA-oligos). Annulus fibrosus (AF) monolayers were poorly responsive to the HA-oligos, proMMP-2 levels were marginally elevated and levels were MMP-9 unaffected. ProMMP-2 displayed a strong dose-dependent increase in the nucleus pulposus (NP) monolayers. In AF alginate bead cultures, proMMP-2 and active MMP-9 increased up to day 10, in NP cultures proMMP-2 was progressively converted to active MMP-2 over days 7-10 and active MMP-9 levels were elevated on day 10. A steady decline in MMP-2 and MMP-9 activity was evident over days 2-10 in the non-stimulated NP cultures. Disc cell viabilities were ≥92 ± 5% in all cultures indicating that the HA-oligo was not cytotoxic. Reverse-transcription polymerase chain reaction demonstrated an upregulation in MMP1, MMP113 and ADAMTS1 and the anabolic matrix repair genes ACAN, COL1A1 and COL2A1 in the NP by HA-oligos, whereas AF MMP13, ADAMTS1, ADAMTS4 and ADAMTS5, ACAN and COL2A1 were down-regulated; this differential regulation is expected to promote clearance of granulation/scar tissue from AF defects and matrix replenishment. The AF defect sites contained enlarged annular lamellae in vivo in response to the HA oligos, which is consistent with an active repair response. Masson trichrome and PicroSirius red histology and immunolocalization of type I collagen supported active remodelling in the outer lesion zone by the HA-oligo treatment but not the inner lesion. Copyright © 2016 John WileySons, Ltd.

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ³⁻/Δ³⁻ exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ³⁻/Δ³⁻ exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell-matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-β a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.

Study design An investigation of mechanical destabilization of the lumbar ovine intervertebral disc (IVD) inducing IVD degeneration (IVDD) as determined by multiparameter outcome measures (magnetic resonance imaging [MRI], IVD composition, biomechanical testing, gene profiling, immunohistochemistry, and immunoblotting). Objective To assess the effect of IVD mechanical destabilization on matrix protein and metalloproteinase gene expression to investigate the pathophysiological mechanisms of lumbar IVDD. Summary of background data Several earlier studies have used annular transection to induce IVDD in sheep, but none have optimized or validated the most appropriate lesion size. Methods The annulus fibrosus (AF) incision inducing maximal change in IVD biomechanics was applied to L1-L2, L3-L4, and L5-L6 discs in vivo to compare with a sham procedure at 3 months post operation. IVDs were evaluated by MRI, biomechanics, histopathology, proteoglycan and collagen content, gene expression, and aggrecan proteolysis by Western blotting. Results Significant changes were observed in lesion (6 × 20 mm(2)) compared with sham IVDs at 3 months post operation: reduced disc height on MRI; increased neutral zone in biomechanical testing; depleted proteoglycan and collagen content in the nucleus pulposus (NP) and lesion half of the AF but not in the contralateral AF; increased messenger RNA for collagen I and II, aggrecan, versican, perlecan, matrix metalloproteinase (MMP)-1 & 13, and ADAMTS-5, in the lesion-site AF and NP but not in the contralateral AF. ADAMTS-4 messenger RNA was increased in the lesion-site AF but decreased in the NP. Despite an upregulation in MMPs, there was no change in MMP- or ADAMTS-generated aggrecan neoepitopes in any region of the IVD in lesion or sham discs. Conclusion Lumbar IVDD was reproducibly induced with a 6 × 20 mm(2) annular lesion, with focal dysregulation of MMP gene expression, cell cloning in the inner AF, loss of NP aggrecan, and disc height. Loss of aggrecan from the NP was not attributable to increased proteolysis in the interglobular domain by MMPs or ADAMTS.

Summary Objective To determine the mechanisms of meniscal degeneration and whether this varied zonally and from articular cartilage. Design Normal ovine menisci were dissected into inner and outer zones and along with cartilage cultured ±IL-1α and TNFα. Glycosaminoglycan (GAG) and collagen release, and gene expression were quantified. Aggrecan proteolysis was analysed by Western blotting with neoepitope-specific antibodies. Matrix metalloproteinase (MMP)2, MMP9 and MMP13 activity was evaluated by gelatin zymography or fluorogenic assay. Results Inner meniscus was more cartilaginous containing more GAG and expressing more ACAN and COL2A1 than outer zones. Higher expression of VCAN and ADAMTS4 in medial outer and both zones of the lateral meniscus reflected their embryologic origin from cells outside the cartilage anlagen. All meniscal regions released a greater % GAG in response to cytokines; only outer zones had cytokine-stimulated collagenolysis. Cytokine-induced aggrecanolysis was primarily due to increased ADAMTS cleavage in cartilage and inner menisci but MMPs in the outer menisci. Outer menisci always released more active MMP2 than other tissues and more active MMP13 in basal and TNF-stimulated cultures. Expression of ACAN , COL1A1 and COL2A1 was decreased by both cytokines in all tissues, while VCAN was increased by IL-1α in cartilage and inner menisci. Metalloproteinase expression was differentially regulated by IL-1α and TNFα: ADAMTS4 , MMP1 , MMP3 were upregulated more by IL-1α in inner zones whereas ADAMTS5 , MMP13 and MMP9 were more upregulated by TNFα in outer zones. Conclusions Meniscal degeneration mechanisms are zonally-dependent, and may contribute to the enzymatic burden in the joint.

Proteoglycans are key components of extracellular matrices, providing structural support as well as influencing cellular behaviour in physiological and pathological processes. The diversity of proteoglycan function reported in the literature is equally matched by diversity in proteoglycan structure. Members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family of enzymes degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular function. In this review, we focus on ADAMTS enzymes that degrade the lectican and small leucine-rich repeat families of proteoglycans. We discuss the known ADAMTS cleavage sites and the consequences of cleavage at these sites. We illustrate our discussion with examples from the literature in which ADAMTS proteolysis of proteoglycans makes profound changes to tissue function.

We undertook a comparative immunolocalisation study on type II collagen, aggrecan and perlecan in a number of 12- to 14-week-old human foetal and postnatal (7-19 months) ovine joints including finger, toe, knee, elbow, hip and shoulder. This demonstrated that perlecan followed a virtually identical immunolocalisation pattern to that of type II collagen in the foetal tissues, but a slightly divergent localisation pattern in adult tissues. Aggrecan was also localised in the cartilaginous joint tissues, which were clearly delineated by toluidine blue staining and the type II collagen immunolocalisations. It was also present in the capsular joint tissues and in ligaments and tendons in the joint, which stained poorly or not at all with toluidine blue. In higher power microscopic views, antibodies to perlecan also stained small blood vessels in the synovial lining tissues of the joint capsule; however, this was not discernable in low power macroscopic views where the immunolocalisation of perlecan to pericellular regions of cells within the cartilaginous rudiments was a predominant feature. Perlecan was also evident in small blood vessels in stromal connective tissues associated with the cartilage rudiments and with occasional nerves in the vicinity of the joint tissues. Perlecan was expressed by rounded cells in the enthesis attachment points of tendons to bone and in rounded cells in the inner third of the meniscus, which stained prominently with type II collagen and aggrecan identifying the chondrogenic background of these cells and local compressive loads. Flattened cells within the tendon and in the surface laminas of articular cartilages and the meniscus did not express perlecan. Collected evidence presented herein, therefore, indicates that besides being a basement membrane component, perlecan is also a marker of chondrogenic cells in prenatal cartilages. In postnatal cartilages, perlecan displayed a pericellular localisation pattern rather than the territorial or interterritorial localisation it displayed in foetal cartilages. This may reflect processing of extracellular perlecan presumably as a consequence of intrinsic biomechanical loading on these tissues or to divergent functions for perlecan and type II collagen in adult compared to prenatal tissues.

The study design included a multidisciplinary examination of the mineral phase of ovine intervertebral disc calcifications. The objective of the study was to investigate the mineral phase and its mechanisms of formation/association with degeneration in a naturally occurring animal model of disc calcification. The aetiology of dystrophic disc calcification in adult humans is unknown, but occurs as a well-described clinical disorder with hydroxyapatite as the single mineral phase. Comparable but age-related pathology in the sheep could serve as a model for the human disorder. Lumbar intervertebral discs (n = 134) of adult sheep of age 6 years (n = 4), 8 years (n = 12) and 11 years (n = 2) were evaluated using radiography, morphology, scanning and transmission electron microscopy, energy dispersive X-ray spectroscopy, X-ray powder diffraction, histology, immunohistology and proteoglycan analysis. Half of the 6-year, 84% of the 8-year and 86% of the 11-year-old discs had calcific deposits. These were not well delineated by plain radiography. They were either: (a) punctate deposits in the outer annulus, (b) diffuse deposits in the transitional zone or inner annulus fibrosus with occasional deposits in the nucleus, or (c) large deposits in the transitional zone extending variably into the nucleus. Their maximal incidence was in the lower lumbar discs (L4/5-L6/7) with no calcification seen in the lumbosacral or lower thoracic discs. All deposits were hydroxyapatite with large crystallite sizes (800-1,300 A) compared to cortical bone (300-600 A). No type X-collagen, osteopontin or osteonectin were detected in calcific deposits, although positive staining for bone sialoprotein was evident. Calcified discs had less proteoglycan of smaller hydrodynamic size than non-calcified discs. Disc calcification in ageing sheep is due to hydroxyapatite deposition. The variable, but large, crystal size and lack of protein markers indicate that this does not occur by an endochondral ossification-like process. The decrease in disc proteoglycan content and size suggests that calcification may precede or predispose to disc degeneration in ageing sheep.

Perlecan is a ubiquitous pericellular proteoglycan ideally placed to mediate cell signaling events controlling migration, proliferation, and differentiation. Its control of growth factor signaling usually involves interactions with the heparan sulfate chains covalently coupled to the protein core's N-terminus. However, this modular protein core also binds with relatively high affinity to a number of growth factors and surface receptors, thereby stabilizing cell-matrix links. This review will focus on perlecan-growth factor interactions and describe recent advances in our understanding of this highly conserved proteoglycan during development, cancer growth, and angiogenesis. The pro-angiogenic capacities of perlecan that involve proliferative and migratory signals in response to bound growth factors will be explored, as well as the anti-angiogenic signals resulting from interactions between the C-terminal domain known as endorepellin and integrins that control adhesion of cells to the extracellular matrix. These two somewhat diametrically opposed roles will be discussed in light of new data emerging from various fields which converge on perlecan as a key regulator of cell growth and angiogenesis.

Objective To evaluate histologic, immunohistochemical, and molecular changes in tendon induced by altered strain in a large-animal model. Methods A full-thickness partial-width laceration of the infraspinatus tendon was created in 5 sheep, while 5 sham-operated sheep were used as controls. Sheep were killed after 4 weeks, and 4 differentially stressed tendon regions (tensile or near bone attachment from overstressed or stress-deprived halves) were evaluated for histopathology, proteoglycan (PG) accumulation, and characterization of glycosaminoglycans and aggrecan catabolites. Gene expression of matrix components, enzymes, and inhibitors was analyzed by reverse transcriptase–polymerase chain reaction. Results Histopathologic changes were detected in both overstressed and stress-deprived tensile tendon, but only in stress-deprived tendon near bone. In overstressed and stress-deprived tensile tendon, levels of keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate were increased. In overstressed tensile tendon, levels of ADAMTS-generated aggrecan catabolites were increased. There was increased matrix metalloproteinase 13 (MMP-13) and decreased fibromodulin and decorin expression in all regions. Increased MMP-1, MMP-9, MMP-14, and ADAMTS-1 expression, and decreased type II collagen expression were restricted to stress-deprived tendon. In stress-deprived bone-attachment regions, messenger RNA (mRNA) for aggrecan was decreased, and ADAMTS was increased. In overstressed tensile tendon, aggrecan mRNA was increased, and ADAMTS was decreased. Conclusion The distinct molecular changes in adjacent tissue implicate altered strain rather than humoral factors in controlling abnormal tenocyte metabolism, and highlight the importance of regional sampling. Tendon abnormalities induced by increased strain are accompanied by increased aggrecan, decreased ADAMTS, and low PG expression, which may negatively impact the structural integrity of the tissue and predispose to rupture.

Intervertebral disc (IVD) degeneration is an often investigated pathophysiological condition because of its implication in causing low back pain. As human material for such studies is difficult to obtain because of ethical and government regulatory restriction, animal tissue, organs and in vivo models have often been used for this purpose. However, there are many differences in cell population, tissue composition, disc and spine anatomy, development, physiology and mechanical properties, between animal species and human. Both naturally occurring and induced degenerative changes may differ significantly from those seen in humans. This paper reviews the many animal models developed for the study of IVD degeneration aetiopathogenesis and treatments thereof. In particular, the limitations and relevance of these models to the human condition are examined, and some general consensus guidelines are presented. Although animal models are invaluable to increase our understanding of disc biology, because of the differences between species, care must be taken when used to study human disc degeneration and much more effort is needed to facilitate research on human disc material.

Perlecan is a ubiquitous proteoglycan of basement membrane and vascularized tissues but is also present in articular cartilage, meniscus and intervertebral disc, which are devoid of basement membrane and predominantly avascular. It is a prominent pericellular proteoglycan in the transitory matrix of the cartilaginous rudiments that develop into components of diarthrodial joints and the axial skeleton, and it forms intricate perichondrial vessel networks that define the presumptive articulating surfaces of developing joints and line the cartilage canals in cartilaginous rudiments. Such vessels have roles in the nutrition of the expanding cell numbers in the developing joint. Perlecan sequesters a number of growth factors pericellularly (FGFs, PDGF, VEGF and CTGF) and through these promotes cell signalling, cell proliferation and differentiation. Perlecan also interacts with a diverse range of extracellular matrix proteins, stabilising and organising the ECM, and promoting collagen fibrillogenesis. Perlecan is a prominent pericellular component of mesenchymal cells from their earliest developmental stages through to maturation, forming cell-cell and cell-ECM interconnections that are suggestive of a role in mechanosensory processes important to tissue homeostasis.

The cartilage growth plate is a specialized developmental tissue containing characteristic zonal arrangements of chondrocytes. The proliferative and differentiative states of chondrocytes are tightly regulated at all stages including the initial limb bud and rudiment cartilage stages of development, the establishment of the primary and secondary ossification centers, development of the growth plates and laying down of bone. A multitude of spatio-temporal signals, including transcription factors, growth factors, morphogens and hormones, control chondrocyte maturation and terminal chondrocyte differentiation/hypertrophy, cell death/differentiation, calcification and vascular invasion of the growth plate and bone formation during morphogenetic transition of the growth plate. This involves hierarchical, integrated signaling from growth and factors, transcription factors, mechanosensory cues and proteases in the extracellular matrix to regulate these developmental processes to facilitate progressive changes in the growth plate culminating in bone formation and endochondral ossification. This review provides an overview of selected components which have particularly important roles in growth plate biology including collagens, proteoglycans, glycosaminoglycans, growth factors, proteases and enzymes.

To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG-2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene-knockout model.Maturational changes were evaluated histologically in the knees of 3-, 6-, and 12-week-old wild-type (WT) mice and Hspg2(Δ3-/Δ3-) mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2(Δ3-/Δ3-) mice. Changes in cartilage expression of FGF-2, FGF-18, HSPG-2, FGF receptor 1 (FGFR-1), and FGFR-3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin-1α (IL-1α), FGF-2, and FGF-18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified.No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2(Δ3-/Δ3-) mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA-induced loss of chondrocyte expression of FGF-2, FGF-18, and HSPG-2 occurred in both genotypes. Expression of FGFR-1 after OA induction was maintained in WT mice, while FGFR-3 loss after OA induction was significantly reduced in Hspg2(Δ3-/Δ3-) mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL-1α-stimulated cartilage. However, IL-1α-induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2(Δ3-/Δ3-) mice. Cartilage GAG release in either the presence or absence of IL-1α was unaltered by FGF-2 in both genotypes. In cartilage cultures with FGF-18, IL-1α-stimulated GAG loss was significantly reduced only in Hspg2(Δ3-/Δ3-) mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA.Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR-3 and increased FGF signaling.

Perlecan is a modular heparan sulphate and/or chondroitin sulphate substituted proteoglycan of basement membrane, vascular tissues and cartilage. Perlecan acts as a low affinity co-receptor for fibroblast growth factors 1, 2, 7, 9, binds connective tissue growth factor and co-ordinates chondrogenesis, endochondral ossification and vascular remodelling during skeletal development; however, relatively little is known of its distribution in these tissues during ageing and development. The aim of the present study was to immunolocalise perlecan in the articular and epiphyseal growth plate cartilages of stifle joints in 2-day to 8-year-old pedigree merino sheep. Perlecan was prominent pericellularly in the stifle joint cartilages at all age points and also present in the inter-territorial matrix of the newborn to 19-month-old cartilage specimens. Aggrecan was part pericellular, but predominantly an extracellular proteoglycan. Perlecan was a prominent component of the long bone growth plates and displayed a pericellular as well as a strong ECM distribution pattern; this may indicate a so far unrecognised role for perlecan in the mineralisation of hypertrophic cartilage. A significant age dependant decline in cell number and perlecan levels was evident in the hyaline and growth plate cartilages. The prominent pericellular distribution of perlecan observed indicates potential roles in cell-matrix communication in cartilage, consistent with growth factor signalling, cellular proliferation and tissue development.

Study design The spatial and temporal localization of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in the injured anulus fibrosus was investigated. Objective To assess the involvement of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in anulus fibrosus repair. Summary of background data Fibroblast growth factor-2 and transforming growth factor-beta have been localized to disc herniation tissue, and alpha-smooth muscle cell actin has been identified in a number of mesenchymal cell types, but their roles have not been evaluated in repair processes in the experimentally injured anulus fibrosus. Methods For this study, 32 two adult merinos received a 4-mm deep standard annular incision in their L1L2 and L3L4 discs (lesion group). A similar number of sham-surgery animals served as control subjects. Osteonectin, fibroblast growth factor-2, transforming growth factor-beta, and alpha-smooth muscle cell actin were immunolocalized in sagittal disc sections 3, 6, 12, and 26 months after the operation. Selected specimens also were stained with hematoxylin and eosin, Masson-trichrome, toluidine blue, and picrosirius red. Results Early focal depletion of proteoglycan was evident in the anulus fibrosus and reorganization of outer annular lamellas 3 to 6 months after the operation. Blood vessel ingrowth and fibroblast infiltration from the outer anulus fibrosus along the plane of the annular defect were maximal 12 months after the operation. Focal upregulation in alpha-smooth muscle cell actin expression was evident with maximal staining in the 12-month lesion samples near infiltrating blood vessels at the lesion site, and also in cells well away from these vessels. Some of the anulus fibrosus cells of the sham sections also stained positively for alpha-smooth muscle cell actin, but this staining was significantly less than in the lesion samples. Staining for fibroblast growth factor-2, transforming growth factor-beta, and osteonectin was strongly localized to blood vessels and cells in the vicinity of the annular lesion. It was maximal 12 months after the operation and diminished by 26 months after the operation. Osteonectin expression also was significantly elevated in outer anulus fibrosus cells distant from the lesion site and its associated blood vessels. In the sham discs, immunoreactivity to fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin was confined to sparsely distributed cells in the anulus fibrosus. No matrix staining was observed. Conclusions Immunoreactivity for the noted agents was strongly associated with regions of the annular lesions undergoing matrix reorganization consistent with an active repair response. This response extended as far as the middle third of the anulus fibrosus, which also demarcated the extent of blood vessel ingrowth and cellular infiltration in this model. The alpha-smooth muscle cell actin expression suggested an active involvement of myofibroblasts in the anulus fibrosus repair processes.

The objective of this study was to determine whether disc cells could be cultured under serum-free conditions and whether they synthesized bovine pancreatic trypsin inhibitor (BPTI)-like serine proteinase inhibitory proteins (SPIs) previously demonstrated for ovine chondrocytes. Intervertebral discs from 1- to 2-year-old merino wether sheep were dissected into the annulus fibrosus and nucleus pulposus, and the cells isolated by sequential enzymatic digestion. The cells were grown encapsulated in calcium alginate microspheres under serum-free conditions for 10 days. They remained more than 92% viable as assessed using the vital fluorescent dyes chloromethyl fluorescein diacetate and ethidium homodimer-1 to delineate live/dead cells, respectively. Western and affinity blotting identified a 12-16-kDa media SPI and an additional 34-36-kDa BPTI-like species in solubilized bead samples. This study has demonstrated ovine disc cells synthesized BPTI-like SPIs in serum-free alginate bead culture similar to chondrocyte SPIs; however, the 58-kDa precursor SPI form was not detected suggesting differences in the endogenous processing of these SPIs.

The aim of this study was to localise hyaluronan (HA)-binding proteins (HABPs) in ovine vertebral tissues using biotinylated HA oligosaccharides (bHA oligos) as novel affinity probes and to compare this with the distribution of tissue HA visualised using biotinylated aggrecan G1 domain-link protein complex. The bHA oligos, with a size of 6-18 disaccharides were prepared by partial digestion of HA with ovine testicular hyaluronidase, labelled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaluronan and HABPs were both prominent pericellular components of hypertrophic cells of the vertebral epiphyseal growth plate and enlarged cells in the cartilaginous end plate of the disc. The bHA oligo probe also visualised HABPs intracellularly in hypertrophic cells, which also contained intracellular HA. Monolayer cultures of ovine annulus fibrosus and nucleus pulposus cells rapidly internalised the bHA oligo affinity probe which was subsequently visualised by indirect fluorescence using avidin-FITC, to cytoplasm and discrete nuclear regions. The results indicate that the abundant pericellular and intracellular HA associated with cartilaginous cells in the vertebral tissues is colocalised with HABPs. The bHA oligo affinity probe may have further applications in investigations of intracellular HABPs, HA endocytosis and the roles they play in cellular regulatory processes.