Higher Glycosaminoglycan Content Research Articles

CNS/PNS proteoglycans functionalize neuronal and astrocyte niche microenvironments optimizing cellular activity by preserving membrane polarization dynamics, ionic microenvironments, ion fluxes, neuronal activation, and network neurotransductive capacity.

Central and peripheral nervous system (CNS/PNS) proteoglycans (PGs) have diverse functional roles, this study examined how these control cellular behavior and tissue function. The CNS/PNS extracellular matrix (ECM) is a dynamic, responsive, highly interactive, space-filling, cell supportive, stabilizing structure maintaining tissue compartments, ionic microenvironments, and microgradients that regulate neuronal activity and maintain the neuron in an optimal ionic microenvironment. The CNS/PNS contains a high glycosaminoglycan content (60% hyaluronan, HA) and a diverse range of stabilizing PGs. Immobilization of HA in brain tissues by HA interactive hyalectan PGs preserves tissue hydration and neuronal activity, a paucity of HA in brain tissues results in a pro-convulsant epileptic phenotype. Diverse CS, KS, and HSPGs stabilize the blood-brain barrier and neurovascular unit, provide smart gel neurotransmitter neuron vesicle storage and delivery, organize the neuromuscular junction basement membrane, and provide motor neuron synaptic plasticity, and photoreceptor and neuron synaptic functions. PG-HA networks maintain ionic fluxes and microgradients and tissue compartments that contribute to membrane polarization dynamics essential to neuronal activation and neurotransduction. Hyalectans form neuroprotective perineuronal nets contributing to synaptic plasticity, memory, and cognitive learning. Sialoglycoprotein associated with cones and rods (SPACRCAN), an HA binding CSPG, stabilizes the inter-photoreceptor ECM. HSPGs pikachurin and eyes shut stabilize the photoreceptor synapse aiding in phototransduction and neurotransduction with retinal bipolar neurons crucial to visual acuity. This is achieved through Laminin G motifs in pikachurin, eyes shut, and neurexins that interact with the dystroglycan-cytoskeleton-ECM-stabilizing synaptic interconnections, neuronal interactive specificity, and co-ordination of regulatory action potentials in neural networks.

Hyaluronan hydrates and compartmentalises the CNS/PNS extracellular matrix and provides niche environments conducive to the optimisation of neuronal activity.

The central nervous system/peripheral nervous system (CNS/PNS) extracellular matrix is a dynamic and highly interactive space-filling, cell-supportive, matrix-stabilising, hydrating entity that creates and maintains tissue compartments to facilitate regional ionic micro-environments and micro-gradients that promote optimal neural cellular activity. The CNS/PNS does not contain large supportive collagenous and elastic fibrillar networks but is dominated by a high glycosaminoglycan content, predominantly hyaluronan (HA) and collagen is restricted to the brain microvasculature, blood-brain barrier, neuromuscular junction and meninges dura, arachnoid and pia mater. Chondroitin sulphate-rich proteoglycans (lecticans) interactive with HA have stabilising roles in perineuronal nets and contribute to neural plasticity, memory and cognitive processes. Hyaluronan also interacts with sialoproteoglycan associated with cones and rods (SPACRCAN) to stabilise the interphotoreceptor matrix and has protective properties that ensure photoreceptor viability and function is maintained. HA also regulates myelination/re-myelination in neural networks. HA fragmentation has been observed in white matter injury, multiple sclerosis, and traumatic brain injury. HA fragments (2 × 105 Da) regulate oligodendrocyte precursor cell maturation, myelination/remyelination, and interact with TLR4 to initiate signalling cascades that mediate myelin basic protein transcription. HA and its fragments have regulatory roles over myelination which ensure high axonal neurotransduction rates are maintained in neural networks. Glioma is a particularly invasive brain tumour with extremely high mortality rates. HA, CD44 and RHAMM (receptor for HA-mediated motility) HA receptors are highly expressed in this tumour. Conventional anti-glioma drug treatments have been largely ineffective and surgical removal is normally not an option. CD44 and RHAMM glioma HA receptors can potentially be used to target gliomas with PEP-1, a cell-penetrating HA-binding peptide. PEP-1 can be conjugated to a therapeutic drug; such drug conjugates have successfully treated dense non-operative tumours in other tissues, therefore similar applications warrant exploration as potential anti-glioma treatments.

Cyclic loading regime considered beneficial does not protect injured and interleukin-1-inflamed cartilage from post-traumatic osteoarthritis

Injurious overloading and inflammation perturbate homeostasis of articular cartilage, leading to abnormal tissue-level loading during post-traumatic osteoarthritis. Our objective was to gain time- and cartilage depth-dependent insights into the early-stage disease progression with an in vitro model incorporating for the first time the coaction of (1) mechanical injury, (2) pro-inflammatory interleukin-1 challenge, and (3) cyclic loading mimicking walking and considered beneficial for cartilage health. Cartilage plugs (n = 406) were harvested from the patellofemoral grooves of young calves (N = 6) and subjected to injurious compression (50% strain, rate 100%/s; INJ), interleukin-1α-challenge (1 ng/ml; IL), and cyclic loading (intermittent 1 h loading periods, 15% strain, 1 Hz; CL). Plugs were assigned to six groups (control, INJ, IL, INJ-IL, IL-CL, INJ-IL-CL). Bulk and localized glycosaminoglycan (GAG) content (DMMB assay, digital densitometry), aggrecan biosynthesis (35S-sulfate incorporation), and chondrocyte viability (fluorescence microscopy) were assessed on days 3–12. The INJ, IL, and INJ-IL groups exhibited rapid early (days 2–4) GAG loss in contrast to CL groups. On day 3, deep cartilage of INJ-IL-CL group had higher GAG content than INJ group (p < 0.05). On day 12, INJ-IL-CL group showed more accumulated GAG loss (normalized with control) than INJ-IL group (average fold changes 1.97 [95% CI: 1.23–2.70]; 1.66 [1.42–1.89]; p = 0.007). Aggrecan biosynthesis increased in CL groups on day 12 compared to day 0. Despite promoting aggrecan biosynthesis, this cyclic loading protocol seems to be beneficial early-on to deep cartilage, but later becoming incapable of restricting further degradation triggered by marked but non-destructive injury and cytokine transport.

A review on 3D printing in tissue engineering applications

Abstract In tissue engineering, 3D printing is an important tool that uses biocompatible materials, cells, and supporting components to fabricate complex 3D printed constructs. This review focuses on the cytocompatibility characteristics of 3D printed constructs, made from different synthetic and natural materials. From the overview of this article, inkjet and extrusion-based 3D printing are widely used methods for fabricating 3D printed scaffolds for tissue engineering. This review highlights that scaffold prepared by both inkjet and extrusion-based 3D printing techniques showed significant impact on cell adherence, proliferation, and differentiation as evidenced by in vitro and in vivo studies. 3D printed constructs with growth factors (FGF-2, TGF-β1, or FGF-2/TGF-β1) enhance extracellular matrix (ECM), collagen I content, and high glycosaminoglycan (GAG) content for cell growth and bone formation. Similarly, the utilization of 3D printing in other tissue engineering applications cannot be belittled. In conclusion, it would be interesting to combine different 3D printing techniques to fabricate future 3D printed constructs for several tissue engineering applications.

Bone Marrow Mesenchymal Stem Cell-Derived Tissues are Mechanically Superior to Meniscus Cells.

Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to form the mechanically responsive matrices of joint tissues, including the menisci of the knee joint. The purpose of this study is to assess BMSC's potential to engineer meniscus-like tissue relative to meniscus fibrochondrocytes (MFCs). MFCs were isolated from castoffs of partial meniscectomy from nonosteoarthritic knees. BMSCs were developed from bone marrow aspirates of the iliac crest. All cells were of human origin. Cells were cultured in type I collagen scaffolds under normoxia (21% O2) for 2 weeks followed by hypoxia (3% O2) for 3 weeks. The structural and functional assessment of the generated meniscus constructs were based on glycosaminoglycan (GAG) content, histological appearance, gene expression, and mechanical properties. The tissues formed by both cell types were histologically positive for Safranin O stain and appeared more intense in the BMSC constructs. This observation was confirmed by a 2.7-fold higher GAG content. However, there was no significant difference in collagen I (COL1A2) expression in BMSC- and MFC-based constructs (p = 0.17). The expression of collagen II (COL2A1) and aggrecan (ACAN) were significantly higher in BMSCs than MFC (p ≤ 0.05). Also, the gene expression of the hypertrophic marker collagen X (COL10A1) was 199-fold higher in BMSCs than MFC (p < 0.001). Moreover, relaxation moduli were significantly higher in BMSC-based constructs at 10-20% strain step than MFC-based constructs. BMSC-based constructs expressed higher COL2A1, ACAN, COL10A1, contained higher GAG content, and exhibited higher relaxation moduli at 10-20% strain than MFC-based construct. Impact statement Cell-based tissue engineering (TE) has the potential to produce functional tissue replacements for irreparably damaged knee meniscus. But the source of cells for the fabrication of the tissue replacements is currently unknown and of research interest in orthopedic TE. In this study, we fabricated tissue-engineered constructs using type I collagen scaffolds and two candidate cell sources in meniscus TE. We compared the mechanical properties of the tissues formed from human meniscus fibrochondrocytes and bone marrow-derived mesenchymal stem cells (BMSCs). Our data show that the tissues engineered from the BMSC are mechanically superior in relaxation modulus.

Tyrosinase-crosslinked, tissue adhesive and biomimetic alginate sulfate hydrogels for cartilage repair

The native cartilage extracellular matrix (ECM) is enriched in sulfated glycosaminoglycans with important roles in the signaling and phenotype of resident chondrocytes. Recapitulating the key ECM components within engineered tissues through biomimicking strategies has potential to improve the regenerative capacity of encapsulated cells and lead to better clinical outcome. Here, we developed a double-modified, biomimetic and tissue adhesive hydrogel for cartilage engineering. We demonstrated sequential modification of alginate with first sulfate moieties to mimic the high glycosaminoglycan content of native cartilage and then tyramine moieties to allow in situ enzymatic crosslinking with tyrosinase under physiological conditions. Tyrosinase-crosslinked alginate sulfate tyramine (ASTA) hydrogels showed strong adhesion to native cartilage tissue with higher bond strength compared to alginate tyramine (AlgTA). Both ASTA and AlgTA hydrogels supported the viability of encapsulated bovine chondrocytes and induced a strong increase in the expression of chondrogenic genes such as collagen 2, aggrecan and Sox9. Aggrecan and Sox9 gene expression of chondrocytes in ASTA hydrogels were significantly higher than those in AlgTA. Chondrocytes in both ASTA and AlgTA hydrogels showed potent deposition of cartilage matrix components collagen 2 and aggrecan after 3 weeks of culture whereas a decreased collagen 1 deposition was observed in the sulfated hydrogels. ASTA and AlgTA hydrogels with encapsulated human chondrocytes showed in vivo stability as well as cartilage matrix deposition upon subcutaneous implantation into mice for 4 weeks. Our data is the first demonstration of a double-modified alginate with sulfation and tyramination that allows in situ enzymatic crosslinking, strong adhesion to native cartilage and chondrogenic re-differentiation.

Hypoxia Preconditioning of Bone Marrow Mesenchymal Stem Cells Before Implantation in Orthopaedics.

Human bone marrow mesenchymal stem cells (hBM-MSCs) have been widely used for tissue engineering applications because of their abundance in adults, relatively easy isolation, ability to differentiate into multiple mesodermal lineages, and capacity to secrete a spectrum of bioactive/therapeutic/trophic factors with chemoattractive, immunomodulatory, angiogenic, antiscarring, and antiapoptotic functions.1–6 Despite the notable advances in the field, their ex vivo expansion remains challenging, often associated with phenotypic drift and senescence, imposing the need for engineering more appropriate/bioinspired in vitro microenvironments to maintain their function and therapeutic potential.7,8 Among the various in vitro microenvironment modulators, physiologically relevant (to the cells) low oxygen tension (frequently termed hypoxia) has been characterized as a critical component of the stem cell niche.9–11 It has even been suggested that hypoxia preconditioning of BM-MSCs should become a clinical standard before their implantation.12 This can be rationalized considering that in vivo tissues and organs undergo a broad range of oxygen tensions, depending on their distance from the capillaries, which is markedly lower than the inhalation oxygen tension (∼20%). After entering the lungs, oxygen travels through the bloodstream, and when it reaches the different organs, oxygen tension ranges from 2% to 9%.13 In the bone marrow, for example, BM-MSCs reside in regions of low oxygen tension, ranging from 1% to 6%.10,11 Herein, the influence of low oxygen tension in hBM-MSC in vitro and in vivo fate, with emphasis to orthopaedic clinical indications, is briefly discussed. Several studies have advocated the beneficial effects of low oxygen tension in hBM-MSCs fate. Hypoxia has been shown to maintain hBM-MSCs undifferentiated state and multipotency,14 and to suppress senescence, typically observed at 20% oxygen tension, via the downregulation of the expression of p16 and extracellular signal regulated kinase.15 In addition, 2% oxygen tension in combination with macromolecular crowding, a biophysical phenomenon known to enhance and accelerate (over 80-fold) extracellular matrix deposition,16–18 resulted in a microenvironment capable of maintaining the phenotype of hBM-MSCs and their multilineage potential.19 It has been reported that the hypoxia-inducible factor-1α is activated under low oxygen conditions, which in turn activates the Twist-related protein and downregulates the cyclin-dependent kinase inhibitor 1 (p21), which stimulates cell proliferation.20 Moreover, hypoxia-activated hypoxia-inducible factor-1α significantly increased hBM-MSCs migration through downregulation of integrin α4 and upregulation of Rho-associated kinase ROCK1 and serine/threonine kinase (Rac1/2/3) pathways.21 Furthermore, hBM-MSCs cultured at 1% oxygen and supplemented with fibroblast growth factor-2 exhibited enhanced cell proliferation, chondrogenesis, osteogenesis, migration, collagen formation and regenerative potential.22 Regarding chondrogenic induction, a wealth of studies has demonstrated the beneficial effect of low oxygen tension. For example, isolation and expansion of hBM-MSCs at 2%23 and 3%24 oxygen tension have been shown to promote chondrogenesis, compared with cells cultured at 20% to 21% oxygen tension, as revealed by the increased expression of chondrogenic-related genes and higher glycosaminoglycan content. Notably, in vivo implantation of hypoxia preconditioning hBM-MSCs embedded in alginate beads enhanced the chondrocyte phenotype of cells, producing a cartilage-like tissue in athymic mice.25 Hypoxia preconditioning of BM-MSCs has also been shown to promote osteogenesis in both in vitro and in vivo settings. Cells cultured at 1% oxygen tension in Arg-Gly-Asp-alginate hydrogels were shown to promote osteogenic and angiogenic responses.26 Two percent oxygen tension has been shown to increase the proliferation and the osteogenic differentiation of stem cells from donors with osteonecrosis of the femoral head, suggesting hypoxia preconditioning as an effective treatment to stimulate bone healing.27 In immunocompromised mice, preconditioned at 1% oxygen tension, as opposed to 20 % oxygen tension, hBM-MSCs that were seeded on hydroxyapatite/tricalcium phosphate-based scaffolds produced soluble and insoluble collagen that brought about a stable extracellular matrix and resulted in improved regenerative potential of the bone.28 Physiologically, for the hBM-MSCs, low oxygen tension has been shown to be beneficial to their proliferation, migration, plasticity, and differentiation in vitro and to augment their therapeutic potential in vivo. Yet again, only a handful of studies have assessed the therapeutic potential of hypoxia preconditioned stem cells in clinic. For example, intracoronary administration of hypoxia preconditioned of hBM-MSCs after acute myocardial infarction has been shown to be safe and feasible.29 It is imperative to conduct more clinical trials to assess the efficacy and efficiency of low oxygen tension-treated hBM-MSCs in orthopaedic clinical indications.

Intervertebral disc swelling maintains strain homeostasis throughout the annulus fibrosus: A finite element analysis of healthy and degenerated discs

Tissues in the intervertebral disc have a large capacity to absorb water, partially due to the high glycosaminoglycan (GAG) content, which decreases linearly from the nucleus pulposus (NP) in the center to the outer annulus. Our recent work showed that fiber network and GAG distribution contributes to development of residual stresses and strains that were compressive in the inner annulus to tensile in the outer annulus. GAG loss in the inner annulus, as observed with early to moderate degeneration, reduced swelling capacity and circumferential-direction stress by over 50%. However, our previous model was not capable of evaluating interactions between the NP and annulus fibrosus (AF) during swelling. In this study, we evaluated the effect of degeneration (GAG content or swelling capacity) on residual stress development throughout the disc. Simulations of moderate to severe degeneration showed a 40% decrease in NP swelling capacity, with a 25% decrease in AF and cartilaginous endplate swelling. Together, these changes in tissue swelling resulted in a decrease in NP pressure (healthy = 0.21 MPa; severe degeneration = 0.03 MPa) that was comparable to observations in human discs. There was a 60% decrease in circumferential-direction residual deformations with early degeneration. Radial-direction stretch switched from compressive to tensile with degeneration, which may increase the risk for tears or delamination. Degeneration had a significant impact on residual stress/stretch and fiber stretch in the posterior AF, which is important for understanding herniation risk. In conclusion, degenerative changes in disc geometry and intradiscal deformations was recreated by only altering NP and AF GAG composition. Since most computational models simulate degeneration by altering material stiffness, this work highlights the importance of directly simulating biochemical composition and distribution to study disc biomechanics with degeneration. Statement of significanceTissues in the intervertebral disc have a large swelling capacity, due to its high glycosaminoglycan content. Our recent work demonstrated the importance of fiber network and glycosaminoglycan distribution residual stresses and strains development. In this study, we evaluated the effect of swelling on intradiscal deformations between the nucleus pulposus and annulus fibrosus. We also investigated the effect of degenerative glycosaminoglycan loss on swelling-based intradiscal deformations of the intact disc and its subcomponents. Decreases in nucleus glycosaminoglycan content resulted in morphological changes observed with degenerated discs and may help to explain mechanisms behind the increases in annular tears and mechanical dysfunction with degeneration.

Intervertebral Disc Swelling Maintains Strain Homeostasis Throughout the Annulus Fibrosus: A Finite Element Analysis of Healthy and Degenerated Discs

Tissues in the intervertebral disc have an excellent capacity to absorb water, largely due to the high glycosaminoglycan (GAG) content, which decreases linearly from the nucleus pulposus (NP) in the center to the outer annulus. Our recent work showed that fiber network and GAG distribution contributes to development of residual stresses and strains that were compressive in the inner annulus to tensile in the outer annulus. GAG loss in the inner annulus, as observed with early to moderate degeneration, reduced swelling capacity and circumferential-direction stress by over 50%. However, our previous model was not capable of evaluating interactions between the NP and annulus fibrosus (AF) during swelling. In this study, we evaluated the effect of degeneration (GAG content or swelling capacity) on residual stress development throughout the disc. Simulations of moderate to severe degeneration showed a 40% decrease in NP swelling capacity, with a 25% decrease in AF and cartilaginous endplate swelling. Together, these changes in tissue swelling resulted in a decrease in NP pressure (healthy = 0.21MPa; severe degeneration = 0.03MPa) that was comparable to observations in human discs. There was a 60% decrease in circumferential-direction residual deformations with early degeneration. Radial-direction stretch switched from compressive to tensile with degeneration, which may increase the risk for tears or delamination. Degeneration had a significant impact on residual stress/stretch and fiber stretch in the posterior AF, which is important for understanding herniation risk. In conclusion, relatively large changes in local strains occurred with degeneration, which may contribute to further degenerative remodeling.

GAG content, fiber stiffness, and fiber angle affect swelling-based residual stress in the intact annulus fibrosus.

Biological tissues with a high glycosaminoglycan (GAG) content have an excellent ability to swell by absorbing water molecules from the surrounding environment. Our recent work showed that anisotropy in tissue swelling depends on the fiber-network architecture, including fiber angle, fiber stiffness, and lamellae structure. However, that work did not evaluate the effect of in situ boundary conditions, such as the kidney-bean shape of the annulus fibrosus (AF), on swelling behavior. The biochemical composition of intact AF is inhomogeneous with respect to GAG composition, collagen fiber angle, and fiber stiffness. Moreover, the GAG content in the inner AF decreases significantly with degeneration. In this study, we investigated the role of GAG content, fiber angle, and fiber stiffness in AF swelling and residual strain development using a finite element model based on a human lumbar disk. Our results showed that the annular ring structure had a great impact on swelling by developing region-dependent compressive stress/stretch in the inner layers and tensile stress/stretch in the outer AF. Swelling-based residual stretch was comparable to experimentally measured values, suggesting an important role of tissue swelling in maintaining residual stresses. Moreover, GAG loss in the inner AF, as observed with degeneration, decreased circumferential-direction stress by over 65%. Homogeneous distributions of fiber angle and stiffness overestimated or underestimated AF swelling behavior, such as swelling ratio, circumferential/axial stretch, and fiber stretch/reorientation. These findings demonstrate the need to include native fiber architecture in finite element models, to accurately predict tissue failure, as well as to cultivate engineered disks.

Advanced glycation products’ levels and mechanical properties of vaginal tissue in pregnancy

ObjectivesNon-enzymatic glycation is closely associated with altered mechanical properties of connective tissue. Pregnancy, marked with high levels of female hormones, confers unique alteration to the mechanical properties of pelvic connective tissues in order to meet their physiological demands. However, there are few studies on glycation content and its influence on the mechanical properties of pelvic connective tissues during pregnancy. We hypothesise that the glycation content in pelvic tissues will change with a corresponding alteration in their mechanical properties, and that these changes are influenced by hormone levels. This study aims to investigate the correlation of vaginal tissue glycation content and mechanical property changes during pregnancy in association with the expression of a key pregnancy hormone (oestrogen) receptor, and an antioxidant enzyme, glyoxalase I. Study designA rat vaginal tissue model (tissues from non-pregnant and E15-E18 (last trimester) pregnant rats) was used in this study. Mechanical characteristics of vaginal tissues were analysed by a ball-indentation technique while modulus and morphology of the collagen fibrils within the tissues were measured with atomic force microscopy. A glycation marker, pentosidine, was quantified by a high performance liquid chromatography. The expression of oestrogen receptor and glyoxalase I in the tissue was qualified by immunochemical staining. The glycosaminoglycan (GAG) concentration difference in the tissues were quantified by a biochemical assay. ResultsPregnant rat vaginal tissue was characterised by significantly lower amounts of pentosidine, higher oestrogen receptor and glyoxalase I expression with larger creep, lower elastic modulus, larger fibril diameter and higher GAG content than their non-pregnant counterpart. There was a negative correlation between pentosidine and vaginal tissue creep. ConclusionThere was a reduction in vaginal tissue pentosidine in pregnancy with an associated increase in oestrogen receptor and glyoxalase I immunoexpression. Reduced glycation was associated with increased creeping of vaginal tissue. Oestrogen may therefore play a role in the increase of the vaginal wall’s capacity to stretch through glyoxalase I up-regulation and subsequent glycation reduction. The new insight of the correlation of women’s oestrogen level, glycation reaction and pelvic tissue mechanical property from this study may enhance our understanding of some pelvic organ diseases.

Effect of platelet-rich plasma on healing tissues in acute ruptured Achilles tendon: a human immunohistochemistry study

BackgroundPlatelet-rich plasma (PRP), an autologous derivative of whole blood that contains a supraphysiological concentration of platelets, is thought to invoke an earlier and improved tissue healing response. This notion has been supported by in-vitro and animal studies in bone, cartilage, tendon, and muscle. To our knowledge no published study exists of the effects of PRP in human tissues in vivo. The aim of our study was to investigate the response of ruptured Achilles tendon treated with PRP. MethodsTendon tissue biopsy samples were obtained from 20 patients with ruptured Achilles tendon by means of ultrasound-guided needle biopsies from the healing area of the Achilles tendon 6 weeks after treatment with PRP or placebo controls (10 patients each). All samples were embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin and alcian blue. Immunohistochemistry markers were used to identify collagen I and III, lymphocytes (CD45), proliferation (KI67), and blood vessels (CD34). All images were masked and analysed with Image J software. FindingsCellularity and glycosaminoglycans content were significantly higher in PRP-treated tendons than in controls (p=0·01 and p<0·001, respectively). Fibre structure of the tissue was significantly better in the PRP group than in the control tissue (p<0·001). Although both groups showed high collagen I staining, content of collagen I was significantly higher in PRP-treated tendons than in control tendons (p=0·0079), whereas collagen III content was not different (p=1·0). The ratio of collagen III to collagen I was significantly lower in PRP samples (p=0·007). There was no significant difference in CD45 expression (p=0·33). However, PRP samples had fewer blood vessels than did control samples (p=0·023). The overall modified Bonar score was significantly lower in PRP samples, which indicates improved early tendon healing. InterpretationThis is the first study, to our knowledge, to report the immunohistochemical response of ruptured human Achilles tendon to PRP. The findings reveal that locally applied PRP enhanced the maturity of the healing tendon tissues by promoting better collagen I deposition, decreased cellularity, less vascularity, and higher glycosaminoglycan content when compared with control samples. Further work is required to determine the longer term effects of the use of PRP in musculoskeletal diseases. FundingNational Institute for Health Research.

The Effect of Osteochondral Regeneration Using Polymer Constructs and Continuous Passive Motion Therapy in the Lower Weight-Bearing Zone of Femoral Trocheal Groove in Rabbits

Remedying patellofemoral osteochondral defects using clinical therapy remains challenging. Construct-based and cell-based regenerative medicine with in vitro physical stimuli has been progressively implemented. However, the effect of physical stimuli in situ in knee joints with degradable constructs is still not well-documented. Therefore, we studied whether it was practical to achieve articular cartilage repair using a poly(lactic-co-glycolic acid) (PLGA) construct in addition to early short-term continuous passive motion (CPM) for treatment of full-thickness osteochondral defects in the lower-weigh bearing (LWB) zone of the femoral trocheal groove. Twenty-six rabbits were randomly allocated into either intermittent active motion (IAM) or CPM treatment groups with or without PLGA constructs, termed PLGA construct-implanted (PCI) and empty defect knee models, respectively. Gross observation, histology, inflammatory cells, which were identified using H&E staining, total collagen and alignment, studied qualitatively using Masson's trichrome staining, glycosaminoglycan (GAG), identified using Alcian blue staining, and newly formed bone, observed using micro-CT, were evaluated at 4 and 12weeks after surgery. Repair of osteochondral defects in the PCI-CPM group was more promising than all other groups. The better osteochondral defect repair in the PCI-CPM group corresponded to smooth cartilage surfaces, no inflammatory reaction, hyaline cartilaginous tissues composition, sound collagen alignment with positive collagen type II expression, higher GAG content, mature bone regeneration with osteocyte, clear tidemark formation, and better degradation of PLGA. In summary, the use of a simple PLGA construct coupled with passive motion promotes positive healing and may be a promising clinical intervention for osteochondral regeneration in LWB defects.

DGEMRIC as a tool for measuring changes in cartilage quality following high tibial osteotomy: a feasibility study

The high tibial osteotomy (HTO) is an effective strategy for treatment of painful medial compartment knee osteoarthritis. Effects on cartilage quality are largely unknown. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) enables non-invasive assessment of cartilage glycosaminoglycan content. This study aimed to evaluate if dGEMRIC could detect relevant changes in cartilage glycosaminoglycan content following HTO. Ten patients with medial compartment osteoarthritis underwent a dGEMRIC scan prior to HTO, and after bone healing and subsequent hardware removal. A dGEMRIC index (T1Gd) was used for changes in cartilage glycosaminoglycan content, a high T1Gd indicating a high glycosaminoglycan content and vice versa. Radiographic analysis included mechanical axis and tibial slope measurement. clinical scores [knee osteoarthritis outcome scale (KOOS), visual analogue score (VAS) for pain, Knee Society clinical rating system (KSCRS)] before, 3 and 6 months after HTO and after hardware removal were correlated to T1Gd changes. Overall a trend towards a decreased T1Gd, despite HTO, was observed. Before and after HTO, lateral femoral condyle T1Gd was higher than medial femoral condyle (MFC) T1Gd and tibial cartilage T1Gd was higher than that of femoral cartilage (P < 0.001). The MFC had the lowest T1Gd before and after HTO. Clinical scores all improved significantly (P < 0.01), KOOS Symptoms and QOL were moderately related to changes in MFC T1Gd. dGEMRIC effectively detected differences in cartilage quality within knee compartments before and after HTO, but no changes due to HTO were detected. Hardware removal post-HTO seems essential for adequate T(1)Gd interpretation. T(1)Gd was correlated to improved clinical scores on a subscore level only. Longer follow-up after HTO may reveal lasting changes. ClinicalTrials.gov registration ID: NCT01269944.

Measurement of glycosaminoglycans in canine synovial fluid and its correlation with the cause of secondary osteoarthritis, age and body weight

Glycosaminoglycans are natural components of healthy joint cartilage and they also appear in healthy synovial fluid. An increased amount of glycosaminoglycans in synovial fluid is believed to be a marker of secondary osteoarthritis, regardless of its primary cause. The aim of our study was to define the relationship between glycosaminoglycans in the synovial fluid and joint disorders, age, and body weight. The samples of synovial fluid were obtained from dogs suffering from secondary secondary osteoarthritis (n = 35) and from control dogs (n = 18); control dogs had normal body weight. The results were compared among joints of dogs with secondary osteoarthritis divided into groups according to the criteria mentioned above and control dogs. Glycosaminoglycan concentrations in synovial fluid were measured using dimethylmethylene blue assay. The lowest mean value of glycosaminoglycans in synovial fluid was measured in the control group. Significantly higher glycosaminoglycan content (P&lt; 0.05) was found in synovial fluid isolated from obese dogs compared to control dogs. Furthermore, we observed an age-related trend, in which the highest mean values were reached either in old dogs or pups. Despite the absence of significant differences in glycosaminoglycan values among dogs suffering from various types of secondary secondary osteoarthritis, the highest mean values were measured in fragmented coronoid processus group. Our data suggest that abnormally increased body weight has an impact on glycosaminoglycan concentration in synovial fluid which may imply faster degradation and turnover of joint cartilage. Such observation has not yet been published in veterinary medicine.

Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation

In this study, we aimed at investigating the interactions between primary chondrocytes and mesenchymal stem/stromal cells (MSC) accounting for improved chondrogenesis in coculture systems. Expanded MSC from human bone marrow (BM-MSC) or adipose tissue (AT-MSC) were cultured in pellets alone (monoculture) or with primary human chondrocytes from articular (AC) or nasal (NC) cartilage (coculture). In order to determine the reached cell number and phenotype, selected pellets were generated by combining: (i) human BM-MSC with bovine AC, (ii) BM-MSC from HLA-A2+ with AC from HLA-A2- donors, or (iii) human green fluorescent protein transduced BM-MSC with AC. Human BM-MSC and AC were also cultured separately in transwells. Resulting tissues and/or isolated cells were assessed immunohistologically, biochemically, cytofluorimetrically, and by RT-PCR. Coculture of NC or AC (25%) with BM-MSC or AT-MSC (75%) in pellets resulted in up to 1.6-fold higher glycosaminoglycan content than what would be expected based on the relative percentages of the different cell types. This effect was not observed in the transwell model. BM-MSC decreased in number (about fivefold) over time and, if cocultured with chondrocytes, increased type II collagen and decreased type X collagen expression. Instead, AC increased in number (4.2-fold) if cocultured with BM-MSC and maintained a differentiated phenotype. Chondro-induction in MSC-chondrocyte coculture is a robust process mediated by two concomitant effects: MSC-induced chondrocyte proliferation and chondrocyte-enhanced MSC chondrogenesis. The identified interactions between progenitor and mature cell populations may lead to the efficient use of freshly harvested chondrocytes for ex vivo cartilage engineering or in situ cartilage repair.

Continuous Gradients of Material Composition and Growth Factors for Effective Regeneration of the Osteochondral Interface

Most contemporary biomaterial designs for osteochondral regeneration utilize monolithic, biphasic, or even multiphasic constructs. We have introduced a microsphere-based approach to create a continuous gradient in both material composition and encapsulated growth factors. The gradients were fabricated by filling a cylindrical mold with opposing gradients of two different types of poly(D,L-lactic-co-glycolic acid) microspheres. The chondrogenic microspheres were loaded with transforming growth factor-β1, whereas the osteogenic microspheres contained bone morphogenetic protein-2 with or without nanophase hydroxyapatite. The gradient scaffolds (material gradient only, signal gradient only, or material/signal gradient combination) or blank control scaffolds were implanted in 3.5 mm-diameter defects in rabbit knees for 6 or 12 weeks. This is the first in vivo evaluation of these novel gradient scaffolds in the knee. The gross morphology, MRI, and histology indicated that the greatest extent of regeneration was achieved when both signal and material gradients were included together. This combination resulted in complete bone ingrowth, with an overlying cartilage layer with high glycosaminoglycan content, appropriate thickness, and integration with the surrounding cartilage and underlying bone. The results suggest that osteochondral regeneration may benefit from biomaterials that integrate a continuous gradient in both material composition and encapsulated growth factors.

Self-assembling peptide hydrogels modulate in vitro chondrogenesis of bovine bone marrow stromal cells.

Our objective was to test the hypothesis that self-assembling peptide hydrogel scaffolds provide cues that enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide hydrogel sequences, and chondrogenesis was compared with that in agarose hydrogels. BMSCs in all three hydrogels underwent transforming growth factor-beta1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of extracellular matrix molecules. Expression of an osteogenic marker was unchanged, and an adipogenic marker was suppressed by transforming growth factor-beta1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide hydrogel sequence, with extensive cell-cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that self-assembling peptide hydrogels enhance chondrogenesis compared with agarose as shown by extracellular matrix production, DNA content, and aggrecan molecular structure.

Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm x 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O(2)) or hypoxic (5% O(2)) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10-20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (+/-25 degrees; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro.

P164 Effects of reduced oxygen tension and long-term mechanical stimulation on chondrocytes-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm x 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O(2)) or hypoxic (5% O(2)) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10-20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (+/-25 degrees; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro.