Sulfated Glycosaminoglycan Content Research Articles

Evaluation of Cartilage-Like Matrix Formation in a Nucleus Pulposus-Derived Cartilage Analog Scaffold.

The formation of fibrocartilage in microfracture (MFX) severely limits its long-term outlook. There is consensus in the scientific community that the placement of an appropriate scaffold in the MFX defect site can promote hyaline cartilage formation and improve therapeutic benefit. Accordingly, in this work, a novel natural biomaterial-the cartilage analog (CA)-which met criteria favorable for chondrogenesis, was evaluated invitro to determine its candidacy as a potential MFX scaffold. Human bone marrow stem cells (hBMSCs) were seeded onto the CA and cultured for 28 days in chondrogenic differentiation media. Sulfated glycosaminoglycan (sGAG) and hydroxyproline (HYP) contents were significantly higher than their non-seeded counterparts on both Days 14 and 28 (average sGAG on Day 28: 73.26 vs. 23.82 μg/mg dry wt. of tissue; average HYP on Day 28: 56.19 vs. 38.80 ± 2.53 μg/mg dry wt. of tissue). Histological assessments showed cellular infiltration and abundant sGAG formation for seeded CAs at both time points with new cartilage-like matrix filling up its laser-drilled channels. Polarized light microscopy of picrosirius red stained samples showed collagen fibrils aligning along the path of the laser-drilled channels. However, the seeded scaffolds were also found to have contracted by 20% by the end of the study with their average aggregate moduli significantly lower than non-seeded controls (10.52 vs. 21.74 kPa). Nevertheless, the CA was ultimately found to support the formation of a cartilage-like matrix, and therefore, merits consideration as a scaffold of interest for improving MFX.

Physiologic Doses of Transforming Growth Factor-β Improve the Composition of Engineered Articular Cartilage.

Conventionally, for cartilage tissue engineering applications, transforming growth factor beta (TGF-β) is administered at doses that are several orders of magnitude higher than those present during native cartilage development. While these doses accelerate extracellular matrix (ECM) biosynthesis, they may also contribute to features detrimental to hyaline cartilage function, including tissue swelling, type I collagen (COL-I) deposition, cellular hypertrophy, and cellular hyperplasia. In contrast, during native cartilage development, chondrocytes are exposed to moderate TGF-β levels, which serve to promote strong biosynthetic enhancements while mitigating risks of pathology associated with TGF-β excesses. Here, we examine the hypothesis that physiologic doses of TGF-β can yield neocartilage with a more hyaline cartilage-like composition and structure relative to conventionally administered supraphysiologic doses. This hypothesis was examined on a model system of reduced-size constructs (∅2 × 2 mm or ∅3 × 2 mm) comprised of bovine chondrocytes encapsulated in agarose, which exhibit mitigated TGF-β spatial gradients allowing for an evaluation of the intrinsic effect of TGF-β doses on tissue development. Reduced-size (∅2 × 2 mm or ∅3 × 2 mm) and conventional-size constructs (∅4-∅6 mm × 2 mm) were subjected to a range of physiologic (0.1, 0.3, 1 ng/mL) and supraphysiologic (3, 10 ng/mL) TGF-β doses. At day 56, the physiologic 0.3 ng/mL dose yielded reduced-size constructs with native cartilage-matched Young's modulus (EY) (630 ± 58 kPa) and sulfated glycosaminoglycan (sGAG) content (5.9 ± 0.6%) while significantly increasing the sGAG-to-collagen ratio, leading to significantly reduced tissue swelling relative to constructs exposed to the supraphysiologic 10 ng/mL TGF-β dose. Furthermore, reduced-size constructs exposed to the 0.3 ng/mL dose exhibited a significant reduction in fibrocartilage-associated COL-I and a 77% reduction in the fraction of chondrocytes present in a clustered morphology, relative to the supraphysiologic 10 ng/mL dose (p < 0.001). EY was significantly lower for conventional-size constructs exposed to physiologic doses due to TGF-β transport limitations in these larger tissues (p < 0.001). Overall, physiologic TGF-β appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating features detrimental to hyaline cartilage function. While reduced-size constructs are not suitable for the repair of clinical-size cartilage lesions, insights from this work can inform TGF-β dosing requirements for emerging scaffold release or nutrient channel delivery platforms capable of achieving uniform delivery of physiologic TGF-β doses to larger constructs required for clinical cartilage repair.

Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism

Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3days prior to collection for biochemical analyses. Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

Characterization and modulation of the pro-inflammatory effects of immune cells in the canine intervertebral disk.

Intervertebral disk (IVD) degeneration affects both humans and canines and is a major cause of low back pain (LBP). Mast cell (MC) and macrophage (MØ) infiltration has been identified in the pathogenesis of IVD degeneration (IVDD) in the human and rodent model but remains understudied in the canine. MC degranulation in the IVD leads to a pro-inflammatory cascade and activates protease activated receptor 2 (PAR2) on IVD cells. The objectives of the present study are to: (1) highlight the pathophysiological changes observed in the degenerate canine IVD, (2) further characterize the inflammatory effect of MCs co-cultured with canine nucleus pulposus (NP) cells, (3) evaluate the effect of construct stiffness on NP and MCs, and (4) identify potential therapeutics to mitigate pathologic changes in the IVD microenvironment. Canine IVD tissue was isolated from healthy autopsy research dogs (beagle) and pet dogs undergoing laminectomy for IVD herniation. Morphology, protein content, and inflammatory markers were assessed. NP cells isolated from healthy autopsy (Mongrel hounds) tissue were co-cultured with canine MCs within agarose constructs and treated with cromolyn sodium (CS) and PAR2 antagonist (PAR2A). Gene expression, sulfated glycosaminoglycan content, and stiffness of constructs were assessed. CD 31+ blood vessels, mast cell tryptase, and macrophage CD 163+ were increased in the degenerate surgical canine tissue compared to healthy autopsy. Pro-inflammatory genes were upregulated when canine NP cells were co-cultured with MCs and the stiffer microenvironment enhanced these effects. Treatment with CS and PAR2 inhibitors mediated key pro-inflammatory markers in canine NP cells. There is increased MC, MØs, and vascular ingrowth in the degenerate canine IVD tissue, similar to observations in the clinical population with IVDD and LBP. MCs co-cultured with canine NP cells drive inflammation, and CS and PAR2A are potential therapeutics that may mitigate the pathophysiology of IVDD in vitro.

Equine bone marrow MSC-derived extracellular vesicles mitigate the inflammatory effects of interleukin-1β on navicular tissues in vitro.

Safe, efficacious therapy for treating degenerate deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) in navicular horses is critically necessary. While archetypal orthobiologic therapies for navicular disease are used empirically, their safety and efficacy are unknown. Mesenchymal stem cell-derived extracellular vesicles (EV) may overcome several limitations of current orthobiologic therapies. To (1) characterise cytokine and growth factor profiles of equine bone marrow mesenchymal stem cell (BM-MSC)-derived extracellular vesicles (BM-EV) and (2) evaluate the in vitro anti-inflammatory and extracellular matrix (ECM) protective potentials of BM-EV on DDFT and NBF explant co-cultures in an IL-1β inflammatory environment. In vitro experimental study. Cytokines (IL-1β, IL-6, IL-10, IL-1ra and TNF-α) and growth factors (TGFβ1, VEGF, IGF1 and PDGF) in equine BM-EV isolated via ultracentrifugation and precipitation methods were profiled. Forelimb DDFT and NBF explant co-cultures from seven horses were exposed to media alone, or media containing 2 × 109 ± 0.1 × 109 particles/mL or 10 μg/mL BM-EV (BM-EV), 10 ng/mL interleukin-1β (IL-1β), or IL-1β + BM-EV for 48 h. Co-culture media IL-6, TNF-α, MMP-3, MMP-13 concentrations and explant sulphated glycosaminoglycan (sGAG) content were quantified. IL-6, IGF1 and VEGF concentrations were 102.1 (37.61-256.2) and 182.3 (163.1-226.3), 72.3 (8-175.6) and 2.4 (0.1-2.6), 108.3 (38.3-709.1) and 211.4 (189.1-318.2) pg/mL per 2 × 109 ± 0.1 × 109 particles/mL or 10 μg/mL 10 μg of BM-EV isolated via ultracentrifugation and precipitation methods, respectively. Co-culture media MMP-3 in BM-EV- (p = 0.03) and BM-EV + IL-1β-treated (p = 0.01) groups were significantly lower than the respective media and IL-1β groups. DDFT explant sGAG content of BM-EV (p = 0.003) and BM-EV + IL-1β groups were significantly higher compared with IL-1β group. Specimen numbers are limited, in vitro model may not replicate clinical case conditions, lack of non-MSC-derived EV control group. Equine BM-EV contains IL-6 and growth factors, IGF1 and VEGF. The anti-inflammatory and ECM protective potentials of BM-EV were evident as increased IL-6 and decreased MMP-3 concentrations in the DDFT-NBF explant co-culture media. These results support further evaluation of BM-EV as an acellular and 'off-the-shelf' intra-bursal/intrasynovial therapy for navicular pathologies.

308 Targeting Cdk8 to improve Ischemic Fracture Healing

OBJECTIVES/GOALS: There are178 million bone fractures globally each year, and 46% of fractures that have accompanying vascular damage (ischemia) will not heal without surgical intervention. Using single cell RNAseq we identified Cdk8 as a gene upregulated under ischemic fracture. Our work seeks to inhibit Cdk8 to assess its potential as a therapeutic target. METHODS/STUDY POPULATION: Most bone injuries heal through a cartilage intermediate that requires mesenchymal progenitor cells (hMSCs) to become cartilage forming chondrocytes. hMSCs underwent pelleted 3D chondrogenic differentiation in the presence of Cdk8 inhibitor, Senexin B. Chondrogenic gene expression was assessed via gene analysis of Aggrecan, Collagen II, and Collagen X. Content of sulfated glycosaminoglycans (sGAGs) was quantified through DMMB analysis. With IACUC approval, C57Bl/6 WT mice underwent femoral artery isolation and resection to create an ischemic environment prior to a transverse tibia fracture. Mice underwent intraperitoneal injections of Senexin B from day -1 to 8 and were harvested at 10 days post fracture. Control mice received vehicle injection. Calluses were analyzed through µCT and histomorphometry. RESULTS/ANTICIPATED RESULTS: At 14 days, Senexin B increased chondrogenic gene expression and improved sGAG content in hMSCs. This persisted to day 21, suggesting that Cdk8 inhibition via Senexin B promotes chondrogenesis and matrix deposition. Histomorphometric analysis reveals thatin vivotreatment with Senexin B increases cartilage content and reduces mineralization of the fracture callus compared to the Control. µCT analysis corroborates this, with distinctly less peri-cortical mineral present in Senexin B-treated calluses, and a decrease in total bone volume. These results suggest an altered progression of cartilage formation and endochondral ossification with Cdk8 inhibition. DISCUSSION/SIGNIFICANCE: Our findings reveal that increased Cdk8 is associated with poor healing in ischemic fractures. Inhibition of Cdk8 appears to increase chondrogenesis of hMSCsin vitroand in the murine fracture callusin vivo. Targeting Cdk8 offers potential to improve callus formation in impaired healing scenarios.

Sodium loading in ambulatory patients with heart failure with reduced ejection fraction: Mechanistic insights into sodium handling.

Sodium restriction was not associated with improved outcomes in heart failure patients in recent trials. The skin might act as a sodium buffer, potentially explaining tolerance to fluctuations in sodium intake without volume overload, but this is insufficiently understood. Therefore, we studied the handling of an increased sodium load in patients with heart failure with reduced ejection fraction (HFrEF). Twenty-one ambulatory, stable HFrEF patients and 10 healthy controls underwent a 2-week run-in phase, followed by a 4-week period of daily 1.2 g (51 mmol) sodium intake increment. Clinical, echocardiographic, 24-h urine collection, and bioelectrical impedance data were collected every 2 weeks. Blood volume, skin sodium content, and skin glycosaminoglycan content were assessed before and after sodium loading. Sodium loading did not significantly affect weight, blood pressure, congestion score, N-terminal pro-brain natriuretic peptide, echocardiographic indices of congestion, or total body water in HFrEF (all p > 0.09). There was no change in total blood volume (4748 ml vs. 4885 ml; p = 0.327). Natriuresis increased from 150 mmol/24 h to 173 mmol/24 h (p = 0.024), while plasma renin decreased from 286 to 88 μU/L (p = 0.002). There were no significant changes in skin sodium content, total glycosaminoglycan content, or sulfated glycosaminoglycan content (all p > 0.265). Healthy controls had no change in volume status, but a higher increase in natriuresis without any change in renin. Selected HFrEF patients can tolerate sodium loading, with increased renal sodium excretion and decreased neurohormonal activation.

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells' natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypicalternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

T1rho MR properties of human patellar cartilage: correlation with indentation stiffness and biochemical contents.

Cartilage degeneration involves structural, compositional, and biomechanical alterations that may be detected non-invasively using quantitative MRI. The goal of this study was to determine if topographical variation in T1rho values correlates with indentation stiffness and biochemical contents of human patellar cartilage. Cadaveric patellae from unilateral knees of 5 donors with moderate degeneration were imaged at 3-Telsa with spiral chopped magnetization preparation T1rho sequence. Indentation testing was performed, followed by biochemical analyses to determine water and sulfated glycosaminoglycan contents. T1rho values were compared to indentation stiffness, using semi-circular regions of interest (ROIs) of varying sizes at each indentation site. ROIs matching the resected tissues were analyzed, and univariate and multivariate regression analyses were performed to compare T1rho values to biochemical contents. Grossly, superficial degenerative change of the cartilage (i.e., roughened texture and erosion) corresponded with regions of high T1rho values. High T1rho values correlated with low indentation stiffness, and the strength of correlation varied slightly with the ROI size. Spatial variations in T1rho values correlated positively with that of the water content (R2 = 0.10, p < 0.05) and negatively with the variations in the GAG content (R2 = 0.13, p < 0.01). Multivariate correlation (R2 = 0.23, p < 0.01) was stronger than either of the univariate correlations. These results demonstrate the sensitivity of T1rho values to spatially varying function and composition of cartilage and that the strength of correlation depends on the method of data analysis and consideration of multiple variables.

Exogenous All-Trans Retinoic Acid Induces Myopia and Alters Scleral Biomechanics in Mice.

Ocular all-trans retinoic acid (atRA) levels are influenced by visual cues, and exogenous atRA has been shown to increase eye size in chickens and guinea pigs. However, it is not clear whether atRA induces myopic axial elongation via scleral changes. Here, we test the hypothesis that exogenous atRA will induce myopia and alter scleral biomechanics in the mouse. Male C57BL/6J mice were trained to voluntarily ingest atRA + vehicle (1% atRA in sugar, 25 mg/kg) (RA: n = 16 animals) or vehicle only (Ctrl: n = 14 animals). Refractive error (RE) and ocular biometry were measured at baseline and after 1 and 2 weeks of daily atRA treatment. Eyes were used in ex vivo assays to measure scleral biomechanics (unconfined compression: n = 18), total scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 23), and specific sGAGs (immunohistochemistry: n = 18). Exogenous atRA caused myopic RE and larger vitreous chamber depth (VCD) to develop by 1 week (RE: -3.7 ± 2.2 diopters [D], P < 0.001; VCD: +20.7 ± 15.1 µm, P < 0.001), becoming more severe by 2 weeks (RE: -5.7 ± 2.2 D, P < 0.001; VCD: +32.3 ± 25.8 µm, P < 0.001). The anterior eye biometry was unaffected. While scleral sGAG content was not measurably affected, scleral biomechanics were significantly altered (tensile stiffness: -30% ± 19.5%, P < 0.001; permeability: +60% ± 95.3%, P < 0.001). In mice, atRA treatment results in an axial myopia phenotype. Eyes developed myopic RE and larger VCD without the anterior eye being affected. The decrease in stiffness and increase in permeability of the sclera are consistent with the form-deprivation myopia phenotype.

Extracellular matrix composition analysis of human articular cartilage for the development of organ-on-a-chip

IntroductionArticular cartilage has a complex extracellular matrix (ECM) that provides it a defined architecture for its load-bearing properties. The complete understanding of ECM components is imperative for developing biomimetic organ-on-a-chip tissue construct. ObjectiveThis study aimed to decellularize and characterize the ECM for its protein profiling to generate a niche for enhanced chondrocyte proliferation. MethodsArticular cartilage scrapings were subjected to mechanical and collagenase digestion, followed by sodium dodecyl sulfate (SDS) treatment for 8 h and 16 h. The de-cellularization efficiency was confirmed by hematoxylin & eosin, alcian blue, masson's trichrome staining, and scanning electron microscopy (SEM). The ECM protein profile was quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a bottom–up approach. ResultsHistological characterization revealed void lacunae that lacked staining for cellular components. The ECM, sulfated glycosaminoglycan content, and collagen fibers were preserved after 8 h and 16 h of de-cellularization. The SEM ultrastructure images showed that few chondrocytes adhered to the ECM after 8 h and cell–free ECM after 16 h of de-cellularization. LC-MS/MS analysis identified 66 proteins with heterotypic collagen types COL1A1-COL6A1, COL14A1, COL22A1 and COL25A1 showed moderate fold change and expression levels, while COL18A1, COL26A1, chondroitin sulfate, matrix metalloproteinase-9 (MMP9), fibronectin, platelet glycoprotein 1 beta alpha (GP1BA), vimentin, bone morphogenetic protein 6 (BMP6), fibroblast growth factor 4 (FGF4) and growth hormone receptor (GHR) showed maximum fold change and expression levels. ConclusionsThe standardized de-cellularization process could preserve majority of ECM components, providing structural integrity and architecture to the ECM. The Identified proteins quantified for their expression levels provided insight into engineering the ECM composition for developing cartilage-on-a-chip.

Co-Culture of Mesenchymal Stem Cells and Ligamentocytes on Triphasic Embroidered Poly(L-lactide-co-ε-caprolactone) and Polylactic Acid Scaffolds for Anterior Cruciate Ligament Enthesis Tissue Engineering

Successful anterior cruciate ligament (ACL) reconstructions strive for a firm bone-ligament integration. With the aim to establish an enthesis-like construct, embroidered functionalized scaffolds were colonized with spheroids of osteogenically differentiated human mesenchymal stem cells (hMSCs) and lapine (l) ACL fibroblasts in this study. These triphasic poly(L-lactide-co-ε-caprolactone) and polylactic acid (P(LA-CL)/PLA) scaffolds with a bone-, a fibrocartilage transition- and a ligament zone were colonized with spheroids directly after assembly (DC) or with 14-day pre-cultured lACL fibroblast and 14-day osteogenically differentiated hMSCs spheroids (=longer pre-cultivation, LC). The scaffolds with co-cultures were cultured for 14 days. Cell vitality, DNA and sulfated glycosaminoglycan (sGAG) contents were determined. The relative gene expressions of collagen types I and X, Mohawk, Tenascin C and runt-related protein (RUNX) 2 were analyzed. Compared to the lACL spheroids, those with hMSCs adhered more rapidly. Vimentin and collagen type I immunoreactivity were mainly detected in the hMSCs colonizing the bone zone. The DNA content was higher in the DC than in LC whereas the sGAG content was higher in LC. The gene expression of ECM components and transcription factors depended on cell type and pre-culturing condition. Zonal colonization of triphasic scaffolds using spheroids is possible, offering a novel approach for enthesis tissue engineering.

Neo-cartilage formation using human nondegenerate versus osteoarthritic chondrocyte-derived cartilage organoids in a viscoelastic hydrogel.

Current regenerative cartilage therapies are associated with several drawbacks such as dedifferentiation of chondrocytes during expansion and the formation of fibrocartilage. Optimized chondrocyte expansion and tissue formation could lead to better clinical results of these therapies. In this study, a novel chondrocyte suspension expansion protocol that includes the addition of porcine notochordal cell-derived matrixwas used to self-assemble human chondrocytes from osteoarthritic (OA) and nondegenerate (ND) origin into cartilage organoids containing collagen type II and proteoglycans. Proliferation rate and viability were similar for OA and ND chondrocytes and organoids formed had a similar histologic appearance and gene expression profile. Organoids were then encapsulated in viscoelastic alginate hydrogels to form larger tissues. Chondrocytes on the outer bounds of the organoids produced a proteoglycan-rich matrix to bridge the space between organoids. In hydrogels containing ND organoids some collagen type I was observed between the organoids. Surrounding the bulk of organoids in the center of the gels, in both OA and ND gels a continuous tissue containing cells, proteoglycans and collagen type II had been produced. No difference was observed in sulphated glycosaminoglycan and hydroxyproline content between gels containing organoids from OA or ND origin after 28 days. It was concluded that OA chondrocytes, which can be harvested from leftover surgery tissue, perform similar to ND chondrocytes in terms of human cartilage organoid formation and matrix production in alginate gels. This opens possibilities for their potential to serve as a platform for cartilage regeneration but also as an in vitro model to study pathways, pathology, or drug development.

Aggrecan governs intervertebral discs development by providing critical mechanical cues of the extracellular matrix.

Aggrecan (ACAN) is localized in the intervertebral disc (IVD) in unique compartment-specific patterns where it contributes to the tissue structure and mechanical function together with collagens. The extracellular matrix (ECM) of the IVD undergoes degenerative changes during aging, misuse or trauma, which inevitably alter the biochemical and biomechanical properties of the tissue. A deeper understanding of these processes can be achieved in genetically engineered mouse models, taking into account the multifaceted aspects of IVD development. In this study, we generated aggrecan insertion mutant mice (Acan iE5/iE5 ) by interrupting exon 5 coding for the G1 domain of ACAN, and analyzed the morphological and mechanical properties of the different IVD compartments during embryonic development. Western blotting using an antibody against the total core protein failed to detect ACAN in cartilage extracts, whereas immunohistochemistry by a G1-specific antibody showed weak signals in vertebral tissues of Acan iE5/iE5 mice. Homozygous mutant mice are perinatally lethal and characterized by short snout, cleft palate and disproportionate dwarfism. Whole-mount skeletal staining and µ-CT analysis of Acan iE5/iE5 mice at embryonic day 18.5 revealed compressed vertebral bodies with accelerated mineralization compared to wild type controls. In Acan iE5/iE5 mice, histochemical staining revealed collapsed extracellular matrix with negligible sulfated glycosaminoglycan content accompanied by a high cellular density. Collagen type II deposition was not impaired in the IVD of Acan iE5/iE5 mice, as shown by immunohistochemistry. Mutant mice developed a severe IVD phenotype with deformed nucleus pulposus and thinned cartilaginous endplates accompanied by a disrupted growth plate structure in the vertebral body. Atomic force microscopy (AFM) imaging demonstrated a denser collagen network with thinner fibrils in the mutant IVD zones compared to wild type. Nanoscale AFM indentation revealed bimodal stiffness distribution attributable to the softer proteoglycan moiety and harder collagenous fibrils of the wild type IVD ECM. In Acan iE5/iE5 mice, loss of aggrecan resulted in a marked shift of the Young's modulus to higher values in all IVD zones. In conclusion, we demonstrated that aggrecan is pivotal for the determination and maintenance of the proper stiffness of IVD and vertebral tissues, which in turn could play an essential role in providing developmental biomechanical cues.

Intradiscal treatment of the cartilage endplate for improving solute transport and disc nutrition.

Poor nutrient transport through the cartilage endplate (CEP) is a key factor in the etiology of intervertebral disc degeneration and may hinder the efficacy of biologic strategies for disc regeneration. Yet, there are currently no treatments for improving nutrient transport through the CEP. In this study we tested whether intradiscal delivery of a matrix-modifying enzyme to the CEP improves solute transport into whole human and bovine discs. Ten human lumbar motion segments harvested from five fresh cadaveric spines (38-66 years old) and nine bovine coccygeal motion segments harvested from three adult steers were treated intradiscally either with collagenase enzyme or control buffer that was loaded in alginate carrier. Motion segments were then incubated for 18h at 37 °C, the bony endplates removed, and the isolated discs were compressed under static (0.2MPa) and cyclic (0.4-0.8 MPa, 0.2Hz) loads while submerged in fluorescein tracer solution (376Da; 0.1mg/ml). Fluorescein concentrations from site-matched nucleus pulposus (NP) samples were compared between discs. CEP samples from each disc were digested and assayed for sulfated glycosaminoglycan (sGAG) and collagen contents. Results showed that enzymatic treatment of the CEP dramatically enhanced small solute transport into the disc. Discs with enzyme-treated CEPs had up to 10.8-fold (human) and 14.0-fold (bovine) higher fluorescein concentration in the NP compared to site-matched locations in discs with buffer-treated CEPs (p < 0.0001). Increases in solute transport were consistent with the effects of enzymatic treatment on CEP composition, which included reductions in sGAG content of 33.5% (human) and 40% (bovine). Whole disc biomechanical behavior-namely, creep strain and disc modulus-was similar between discs with enzyme- and buffer-treated CEPs. Taken together, these findings demonstrate the potential for matrix modification of the CEP to improve the transport of small solutes into whole intact discs.

Macromolecular crowding and decellularization method increase the growth factor binding potential of cell-secreted extracellular matrices.

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner.

Development of Brain-Derived Bioscaffolds for Neural Progenitor Cell Culture.

Biomaterials derived from brain extracellular matrix (ECM) have the potential to promote neural tissue regeneration by providing instructive cues that can direct cell survival, proliferation, and differentiation. This study focused on the development and characterization of microcarriers derived from decellularized brain tissue (DBT) as a platform for neural progenitor cell culture. First, a novel detergent-free decellularization protocol was established that effectively reduced the cellular content of porcine and rat brains, with a >97% decrease in the dsDNA content, while preserving collagens (COLs) and glycosaminoglycans (GAGs). Next, electrospraying methods were applied to generate ECM-derived microcarriers incorporating the porcine DBT that were stable without chemical cross-linking, along with control microcarriers fabricated from commercially sourced bovine tendon COL. The DBT microcarriers were structurally and biomechanically similar to the COL microcarriers, but compositionally distinct, containing a broader range of COL types and higher sulfated GAG content. Finally, we compared the growth, phenotype, and neurotrophic factor gene expression levels of rat brain-derived progenitor cells (BDPCs) cultured on the DBT or COL microcarriers within spinner flask bioreactors over 2 weeks. Both microcarrier types supported BDPC attachment and expansion, with immunofluorescence staining results suggesting that the culture conditions promoted BDPC differentiation toward the oligodendrocyte lineage, which may be favorable for cell therapies targeting remyelination. Overall, our findings support the further investigation of the ECM-derived microcarriers as a platform for neural cell derivation for applications in regenerative medicine.

Characteristics of sulfated glycosaminoglycans in the cartilage tissue of skate and sturgeon

Sulfated glycosaminoglycans (GAGs) or chondroitin sulfates were obtained from the cartilage tissue of skate (indefinite species) and sturgeon (kaluga) under the processes of hydrothermal treatment of raw materials at 50 о C, then hydrolysis with proteolytic enzyme in 4 hours under temperature 45 о C (hydromodulus 1 : 1), then sequential precipitation of GAGs in 2–3 volumes of 96 о ethyl alcohol and salting out with 2 % sodium chloride solution. The content of hexosamines and sulfated GAGs was determined in the extracted samples. GAGs of both skate and sturgeon have a high content of sulfated forms, comparable to commercial preparations, that opens up prospects for development of technology for production of monocomponent biologically active additives of chondroprotective action from the wastes of skates and sturgeons processing.

Altered Structure and Function of Murine Sclera in Form-Deprivation Myopia.

The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process. Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE+choroid+sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes). Significant myopia developed after 1 (-2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (-4.1 ± 0.7 D, P=0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = -31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week. We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

Smooth muscle matrix bioink promotes myogenic differentiation of encapsulated adipose-derived stem cells.

Hydrogels derived from decellularized extracellular matrices (dECM) can mimic the biochemical composition of the native tissue. They can also act as a template to culture reseeded cells in vitro. However, detergent-based decellularization methods are known to alter the biochemical compositions, thereby compromising the bioactive potential of dECM. This study proposes a facile detergent-free method to achieve dECM from smooth muscle tissue. We have used the muscle layer of caprine esophageal tissue and decellularized using hypo and hyper-molar sodium chloride solutions alternatingly. Then, a hydrogel was prepared from this decellularized smooth muscle matrix (dSMM) and characterized thoroughly. A comparative analysis of the dSMM prepared with our protocol with the existing detergent-based protocol suggests successful and comparable decellularization with minimal residual DNA content. Interestingly, an 8.78-fold increase in sulfated glycosaminoglycans content and 1.62-fold increased collagen content indicated higher retention of ECM constituents with NaCl-based decellularization strategy. Moreover, the dSMM gel induces differentiation of the encapsulated adipose-derived mesenchymal stem cells toward smooth muscle cells (SMCs) as observed by their expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, the hallmarks of SMCs. Finally, we optimized the process parameter for productive bioprinting with this dSMM bioink and fabricated 3D muscle constructs. Our results suggest that dSMM has the potential to be used as a bioink to engineer personalized esophageal tissues.