Microdialysis Reveals Anti-Inflammatory Effects of Sulfated Glycosaminoglycanes in the Early Phase of Bone Healing.

Although chronic inflammation inhibits bone healing, the healing process is initiated by an inflammatory phase. In a well-tuned sequence of molecular events, pro-inflammatory cytokines are secreted to orchestrate the inflammation response to injury and the recruitment of progenitor cells. These events in turn activate the secretion of anti-inflammatory signaling molecules and attract cells and mediators that antagonize the inflammation and initiate the repair phase. Sulfated glycosaminoglycanes (sGAG) are known to interact with cytokines, chemokines and growth factors and, thus, alter the availability, duration and impact of those mediators on the local molecular level. sGAG-coated polycaprolactone-co-lactide (PCL) scaffolds were inserted into critical-size femur defects in adult male Wistar rats. The femur was stabilized with a plate, and the defect was filled with either sGAG-containing PCL scaffolds or autologous bone (positive control). Wound fluid samples obtained by microdialysis were characterized regarding alterations of cytokine concentrations over the first 24 h after surgery. The analyses revealed the inhibition of the pro-inflammatory cytokines IL-1ß and MIP-2 in the sGAG-treated groups compared to the positive control. A simultaneous increase of IL-6 and TNF-α indicated advanced regenerative capacity of sGAG, suggesting their potential to improve bone healing.

Impact of Sulfated Hyaluronan on Bone Metabolism in Diabetic Charcot Neuroarthropathy and Degenerative Arthritis.

Bone in diabetes mellitus is characterized by an altered microarchitecture caused by abnormal metabolism of bone cells. Together with diabetic neuropathy, this is associated with serious complications including impaired bone healing culminating in complicated fractures and dislocations, especially in the lower extremities, so-called Charcot neuroarthropathy (CN). The underlying mechanisms are not yet fully understood, and treatment of CN is challenging. Several in vitro and in vivo investigations have suggested positive effects on bone regeneration by modifying biomaterials with sulfated glycosaminoglycans (sGAG). Recent findings described a beneficial effect of sGAG for bone healing in diabetic animal models compared to healthy animals. We therefore aimed at studying the effects of low- and high-sulfated hyaluronan derivatives on osteoclast markers as well as gene expression patterns of osteoclasts and osteoblasts from patients with diabetic CN compared to non-diabetic patients with arthritis at the foot and ankle. Exposure to sulfated hyaluronan (sHA) derivatives reduced the exaggerated calcium phosphate resorption as well as the expression of genes associated with bone resorption in both groups, but more pronounced in patients with CN. Moreover, sHA derivatives reduced the release of pro-inflammatory cytokines in osteoclasts of patients with CN. The effects of sHA on osteoblasts differed only marginally between patients with CN and non-diabetic patients with arthritis. These results suggest balancing effects of sHA on osteoclastic bone resorption parameters in diabetes.

Chemically modified glycosaminoglycan derivatives as building blocks for biomaterial coatings and hydrogels.

Tissue regeneration is regulated by the cellular microenvironment, e.g. the extracellular matrix. Here, sulfated glycosaminoglycans (GAG), are of vital importance interacting with mediator proteins and influencing their biological activity. Hence, they are promising candidates for controlling tissue regeneration. This review addresses recent achievements regarding chemically modified GAG as well as collagen/GAG-based coatings and hydrogels including (i) chemical functionalization strategies for native GAG, (ii) GAG-based biomaterial strategies for controlling cellular responses, (iii) (bio)chemical methods for characterization and iv) protein interaction profiles and attained tissue regeneration in vitro and in vivo. The potential of GAG for bioinspired, functional biomaterials is highlighted.

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Artificial Extracellular Matrices Containing Bioactive Glass Nanoparticles Promote Osteogenic Differentiation in Human Mesenchymal Stem Cells.

The present study analyzes the capacity of collagen (coll)/sulfated glycosaminoglycan (sGAG)-based surface coatings containing bioactive glass nanoparticles (BGN) in promoting the osteogenic differentiation of human mesenchymal stroma cells (hMSC). Physicochemical characteristics of these coatings and their effects on proliferation and osteogenic differentiation of hMSC were investigated. BGN were stably incorporated into the artificial extracellular matrices (aECM). Oscillatory rheology showed predominantly elastic, gel-like properties of the coatings. The complex viscosity increased depending on the GAG component and was further elevated by adding BGN. BGN-containing aECM showed a release of silicon ions as well as an uptake of calcium ions. hMSC were able to proliferate on coll and coll/sGAG coatings, while cellular growth was delayed on aECM containing BGN. However, a stimulating effect of BGN on ALP activity and calcium deposition was shown. Furthermore, a synergistic effect of sGAG and BGN was found for some donors. Our findings demonstrated the promising potential of aECM and BGN combinations in promoting bone regeneration. Still, future work is required to further optimize the BGN/aECM combination for increasing its combined osteogenic effect.

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans.

Many viruses, including herpes simplex (HSV), are recruited to their host cells via interaction between their envelope glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial attachment is of a multivalent nature, i.e., it requires the establishment of multiple bonds between amino acids of viral glycoproteins and sulfated saccharides on the GAG chain. To gain understanding of how this binding process is modulated, we performed binding kinetics and mobility studies using end-grafted GAG chains that mimic the end attachment of these chains to proteoglycans. Total internal reflection fluorescence microscopy was used to probe binding and release, as well as the diffusion of single HSV-1 particles. To verify the hypothesis that the degree of sulfation, but also the arrangement of sulfate groups along the GAG chain, plays a key role in HSV binding, we tested two native GAGs (chondroitin sulfate and heparan sulfate) and compared our results to chemically sulfated hyaluronan. HSV-1 recognized all sulfated GAGs, but not the nonsulfated hyaluronan, indicating that binding is specific to the presence of sulfate groups. Furthermore we observed that a notable fraction of GAG-bound virions exhibit lateral mobility, although the multivalent binding to the immobilized GAG brushes ensures firm virus attachment to the interface. Diffusion was faster on the two native GAGs, one of which, chondroitin sulfate, was also characterized by the highest association rate per GAG chain. This highlights the complexity of multivalent virus-GAG interactions and suggests that the spatial arrangement of sulfates along native GAG chains may play a role in modulating the characteristics of the HSV-GAG interaction. Altogether, these results, obtained with a minimal and well-controlled model of the cell membrane, provide, to our knowledge, new insights into the dynamics of the HSV-GAG interaction.

Sulfated hyaluronan attenuates inflammatory signaling pathways in macrophages involving induction of antioxidants.

It is well recognized that high molecular weight hyaluronan (H-HA) exerts potent anti-inflammatory effects while its fragmentation into low molecular weight HA (L-HA) is discussed to promote inflammation. Chemical modification of HA with sulfate groups has been shown to foster its anti-inflammatory activity which seems to be maintained in sulfated low molecular weight HA derivatives (sL-HA). However, the molecular mechanisms by which sL-HA produces its anti-inflammatory activity are not understood. In this study, we used global quantitative proteomics combined with targeted analysis of key proteins to characterize the effect of sL-HA on fully differentiated human inflammatory macrophages (iMФ). Culture of iMФ with sL-HA did not affect cell viability but resulted in a reduced pro-inflammatory cytokine response of iMФ after activation indicating a profound counter-regulation of their initial inflammatory phenotype. Rapid internalization of sL-HA involving CD44 and scavenger receptors was observed. Furthermore, an upregulation of the antioxidants SOD2 and SOD3 was found while no oxidative stress was induced. Consequently, activity of transcription factors for inflammatory gene expression was downregulated in iMФ with sL-HA after activation whereas anti-inflammatory proteins were induced. This study proves anti-inflammatory properties of sL-HA and provides information on its regulatory mode of action on iMФ.

Dual-Functional Hydrazide-Reactive and Anhydride-Containing Oligomeric Hydrogel Building Blocks.

Biomimetic hydrogels are advanced biomaterials that have been developed following different synthetic routes. Covalent postfabrication functionalization is a promising strategy to achieve efficient matrix modification decoupled of general material properties. To this end, dual-functional macromers were synthesized by free radical polymerization of maleic anhydride with diacetone acrylamide (N-(1,1-dimethyl-3-oxobutyl)acrylamide) and pentaerythritol diacrylate monostearate. Amphiphilic oligomers (Mn < 7.5 kDa) with anhydride contents of 7-20% offered cross-linking reactivity to yield rigid hydrogels with gelatinous peptides (E = 4-13 kPa) and good cell adhesion properties. Mildly reactive methyl ketones as second functionality remained intact during hydrogel formation and potential of covalent matrix modification was shown using hydrazide and hydrazine model compounds. Successful secondary dihydrazide cross-linking was demonstrated by an increase of hydrogel stiffness (>40%). Efficient hydrazide/hydrazine immobilization depending on solution pH, hydrogel ketone content as well as ligand concentration for bioconjugation was shown and reversibility of hydrazone formation was indicated by physiologically relevant hydrazide release over 7 days. Proof-of-concept experiments with hydrazido-functionalized hyaluronan demonstrated potential for covalent aECM immobilization. The presented dual-functional macromers have perspective as reactive hydrogel building blocks for various biomedical applications.

Artificial matrices with high-sulfated glycosaminoglycans and collagen are anti-inflammatory and pro-osteogenic for human mesenchymal stromal cells.

Bone healing has been described to be most efficient if the early inflammatory phase is resolved timely. When the inflammation elevates or is permanently established, bone healing becomes impaired and, moreover, bone destruction often takes place. Systemic disorders such as diabetes and bone diseases like arthritis and osteoporosis are associated with sustained inflammation and delayed bone healing. One goal of biomaterial research is the development of materials/surface modifications which support the healing process by inhibiting the inflammatory bone erosion and suppressing pro-inflammatory mediators and by that promoting the bone repair process. In the present study, the influence of artificial extracellular matrices (aECM) on the interleukin (IL)-1ß-induced pro-inflammatory response of human mesenchymal stromal cells (hMSC) was studied. hMSC cultured on aECM composed of collagen I and high-sulfated glycosaminoglycan (GAG) derivatives did not secrete IL-6, IL-8, monocyte chemoattractant protein-1, and prostaglandin E2 in response to IL-1ß. The activation and nuclear translocation of nuclear factor κBp65 induced by IL-1ß, tumor necrosis factor-α or lipopolysaccharide was abrogated. Furthermore, these aECM promoted the osteogenic differentiation of hMSC as determined by an increased activity of tissue non-specific alkaline phosphatase (TNAP); however, the aECM had no effect on the IL-1ß-induced TNAP activity. These data suggest that aECM with high-sulfated GAG derivatives suppress the formation of pro-inflammatory mediators and simultaneously promote the osteogenic differentiation of hMSC. Therefore, these aECM might offer an interesting approach as material/surface modification supporting the bone healing process.

Coating with artificial matrices from collagen and sulfated hyaluronan influences the osseointegration of dental implants.

Dental implants are an established therapy for oral rehabilitation. High success rates are achieved in healthy bone, however, these rates decrease in compromised host bone. Coating of dental implants with components of the extracellular matrix is a promising approach to enhance osseointegration in compromised peri-implant bone. Dental titanium implants were coated with an artificial extracellular matrix (aECM) consisting of collagen type I and either one of two regioselectively low sulfated hyaluronan (sHA) derivatives (coll/sHA1Δ6s and coll/sHA1) and compared to commercial pure titanium implants (control). After extraction of the premolar teeth, 36 implants were inserted into the maxilla of 6 miniature pigs (6 implants per maxilla). The healing periods were 4 and 8 weeks, respectively. After animal sacrifice, the samples were evaluated histomorphologically and histomorphometrically. All surface states led to a sufficient implant osseointegration after 4 and 8 weeks. Inflammatory or foreign body reactions could not be observed. After 4 weeks of healing, implants coated with coll/sHA1Δ6s showed the highest bone implant contact (BIC; coll/sHA1Δ6s: 45.4%; coll/sHA1: 42.2%; control: 42.3%). After 8 weeks, a decrease of BIC could be observed for coll/sHA1Δ6s and controls (coll/sHA1Δ6s: 37.3%; control: 31.7 %). For implants coated with coll/sHA1, the bone implant contact increased (coll/sHA1: 50.8%). Statistically significant differences could not be observed. Within the limits of the current study, aECM coatings containing low sHA increase peri-implant bone formation around dental implants in maxillary bone compared to controls in the early healing period.

Collective pedagogical teacher culture, teacher-student ethno-racial mismatch, and teacher job satisfaction.

Teacher job satisfaction is critical to schools' successful functioning. Using a representative sample of kindergarten teachers from the Early Childhood Longitudinal Study, we investigate the association among professional learning community and teacher collaboration, teacher ethno-racial group, teacher-student ethno-racial mismatch, and teacher job satisfaction. We find that White teachers are significantly less satisfied than African-American and Latino teachers, especially when they teach in majority non-White classrooms. However, the existence of a professional community moderates the negative influence of teacher-student ethno-racial mismatch on White teachers' job satisfaction. In effect, strong professional communities serve as a cushion to bolster teacher job satisfaction.

Do working parents in the United States expect work location to impact job and family satisfaction in the post-pandemic period? Evidence from a survey experiment.

The pandemic response allowed many parents in the United States and globally to work remotely for the first time ever which, for many, continued into the recovery. It is unclear whether, after a period when a large segment of the United States labor force worked remotely, remote work is viewed favorably or unfavorably among employed parents. We present results from a survey experiment assessing whether employed parents in the United States perceive that remote work will impact a hypothetical employed parents' job and family satisfaction and, critically, whether perceptions of work-family conflict and anticipated job rewards mediate this relationship. We find that respondents who are also employed parents perceive that hypothetical employed parents who access remote work will report lower job satisfaction and higher family satisfaction. Perceptions of work-family conflict do not mediate this association. Rather, we find that job rewards (e.g., pay, promotion, etc.) fully mediate the relationship between remote work and perceived job satisfaction. Ultimately, this indicates that employed parents perceive that remote work will bring workers like them less pay and thus lower job satisfaction but greater family satisfaction. This extends arguments about remote work in the light of the conceptualization of a flexibility stigma and a flexibility paradox. Implications for practice and theory are discussed.

Sulfated hyaluronan containing collagen matrices enhance cell-matrix-interaction, endocytosis, and osteogenic differentiation of human mesenchymal stromal cells.

Inorganic-organic composite implant materials mimicking the environment of bone are promising applications to meet the increasing demands on biomaterials for bone regeneration caused by extended life spans and the concomitant increase of bone treatments. Besides collagen type I (Col-I) glycosaminoglycans (GAG), such as hyaluronan, are important components of the bone extracellular matrix (ECM). Sulfated GAGs are potential stimulators of bone anabolic activity, as they are involved in the recruitment of mesenchymal stromal cells (MSCs) to the site of bone formation and support differentiation to osteoblasts. Nevertheless, no consecutive data is currently available about the interaction of hyaluronan or sulfated hyaluronan derivatives with hMSCs and the molecular processes being consequently regulated. We applied quantitative proteomics to investigate the influence of artificial ECM composed of Col-I and hyaluronan (Hya) or sulfated hyaluronan (HyaS3) on the molecular adaptation of osteogenic-differentiated human MSCs (hMSCs). Of the 1,370 quantified proteins, the expression of 4-11% was altered due to both aECM-combinations. Our results indicate that HyaS3 enhanced multiple cell functions, including cell-matrix-interaction, cell-signaling, endocytosis, and differentiation. In conclusion, this study provides fundamental insights into regulative cellular responses associated with HyaS3 and Hya as components of aECM and underlines the potential of HyaS3 as a promising implant-coating-material.

Over-sulfated chondroitin sulfate derivatives induce osteogenic differentiation of hMSC independent of BMP-2 and TGF-ß1 signalling.

Natural glycosaminoglycans (GAGs) and chemically modified GAG derivatives are known to support osteogenic differentiation of mesenchymal stromal cells (MSC). This effect has mainly been described to be mediated by increasing the effectiveness of bone anabolic growth factors such as bone morphogenetic proteins (BMPs) due to the binding and presentation of the growth factor or by modulating its signal transduction pathway. In the present study, the influence of chondroitin sulfate (CS) and two chemically over-sulfated CS derivatives on osteogenic differentiation of human mesenchymal stromal cells (hMSC) and on BMP-2 and transforming growth factor ß1 (TGF-ß1) signalling was investigated. Over-sulfated CS derivatives induced an increase of tissue non-specific alkaline phosphatase (TNAP) activity and calcium deposition, whereas collagen synthesis was slightly decreased. The BMP-2-induced Smad1/5 activation was inhibited in the presence of over-sulfated CS derivatives leading to a loss of BMP-2-induced TNAP activity and calcium deposition. In contrast, the TGF-ß1-induced activation of Smad2/3 and collagen synthesis were not affected by the over-sulfated CS derivatives. BMP-2 and TGF-ß1 did not activate the extracellular signal-regulated kinase 1/2 or mitogen-activated protein kinase p38 in hMSC. These data suggest that over-sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP-2 and TGF-ß1 signalling, and offer therefore an interesting approach for the improvement of bone healing.

Ketoprofen-Based Polymer-Drug Nanoparticles Provide Anti-Inflammatory Properties to HA/Collagen Hydrogels.

Current limitations of wound dressings for treating chronic wounds require the development of novel approaches. One of these is the immune-centered approach, which aims to restore the pro-regenerative and anti-inflammatory properties of macrophages. Under inflammatory conditions, ketoprofen nanoparticles (KT NPs) can reduce pro-inflammatory markers of macrophages and increase anti-inflammatory cytokines. To assess their suitability as part of wound dressings, these NPs were combined with hyaluronan (HA)/collagen-based hydro- (HGs) and cryogels (CGs). Different HA and NP concentrations and loading techniques for NP incorporation were used. The NP release, gel morphology, and mechanical properties were studied. Generally, colonialization of the gels with macrophages resulted in high cell viability and proliferation. Furthermore, direct contact of the NPs to the cells reduced the level of nitric oxide (NO). The formation of multinucleated cells on the gels was low and further decreased by the NPs. For the HGs that produced the highest reduction in NO, extended ELISA studies showed reduced levels of the pro-inflammatory markers PGE2, IL-12 p40, TNF-α, and IL-6. Thus, HA/collagen-based gels containing KT NPs may represent a novel therapeutic approach for treating chronic wounds. Whether effects observed in vitro translate into a favorable profile on skin regeneration in vivo will require rigorous testing.

High-Sulfated Glycosaminoglycans Prevent Coronavirus Replication.

Coronaviruses (CoVs) are common among humans and many animals, causing respiratory or gastrointestinal diseases. Currently, only a few antiviral drugs against CoVs are available. Especially for SARS-CoV-2, new compounds for treatment of COVID-19 are urgently needed. In this study, we characterize the antiviral effects of two high-sulfated glycosaminoglycan (GAG) derivatives against SARS-CoV-2 and bovine coronaviruses (BCoV), which are both members of the Betacoronavirus genus. The investigated compounds are based on hyaluronan (HA) and chondroitin sulfate (CS) and exhibit a strong inhibitory effect against both CoVs. Yield assays were performed using BCoV-infected PT cells in the presence and absence of the compounds. While the high-sulfated HA (sHA3) led to an inhibition of viral growth early after infection, high-sulfated CS (sCS3) had a slightly smaller effect. Time of addition assays, where sHA3 and sCS3 were added to PT cells before, during or after infection, demonstrated an inhibitory effect during all phases of infection, whereas sHA3 showed a stronger effect even after virus absorbance. Furthermore, attachment analyses with prechilled PT cells revealed that virus attachment is not blocked. In addition, sHA3 and sCS3 inactivated BCoV by stable binding. Analysis by quantitative real-time RT PCR underlines the high potency of the inhibitors against BCoV, as well as B.1-lineage, Alpha and Beta SARS-CoV-2 viruses. Taken together, these results demonstrated that the two high-sulfated GAG derivatives exhibit low cytotoxicity and represent promising candidates for an anti-CoV therapy.

Sulfated hyaluronic acid inhibits the hyaluronidase CEMIP and regulates the HA metabolism, proliferation and differentiation of fibroblasts.

Hyaluronan (HA) is an extracellular matrix component that regulates a variety of physiological and pathological processes. The function of HA depends both on its overall amount and on its size, properties that are controlled by HA synthesizing and degrading enzymes. The lack of inhibitors that can specifically block individual HA degrading enzymes has hampered attempts to understand the contribution of individual hyaluronidases to different physiological and pathological processes. CEMIP is a recently discovered hyaluronidase that cleaves HA through mechanisms and under conditions that are distinct from those of other hyaluronidases such as HYAL1 or HYAL2. The role of its hyaluronidase activity in physiology and disease is poorly understood. Here, we characterized a series of sulfated HA derivatives (sHA) with different sizes and degrees of sulfation for their ability to inhibit specific hyaluronidases. We found that highly sulfated sHA derivatives potently inhibited CEMIP hyaluronidase activity. One of these compounds, designated here as sHA3.7, was characterized further and shown to inhibit CEMIP with considerable selectivity over other hyaluronidases. Inhibition of CEMIP with sHA3.7 in fibroblasts, which are the main producers of HA in the interstitial matrix, increased the cellular levels of total and high molecular weight HA, while decreasing the fraction of low molecular weight HA fragments. Genetic deletion of CEMIP in mouse embryonic fibroblasts (MEFs) produced analogous results and confirmed that the effects of sHA3.7 on HA levels were mediated by CEMIP inhibition. Importantly, both CEMIP deletion and its inhibition by sHA3.7 suppressed fibroblast proliferation, while promoting differentiation into myofibroblasts, as reflected in a lack of CEMIP in myofibroblasts within skin wounds in experimental mice. By contrast, adipogenic and osteogenic differentiation were attenuated upon CEMIP loss or inhibition. Our results demonstrate the importance of CEMIP for the HA metabolism, proliferation and differentiation of fibroblasts, and suggest that inhibition of CEMIP with sulfated HA derivatives such as sHA3.7 has potential utility in pathological conditions that are dependent on CEMIP function.

Sulfated glycosaminoglycans inhibit transglutaminase 2 by stabilizing its closed conformation.

Transglutaminases (TGs) catalyze the covalent crosslinking of proteins via isopeptide bonds. The most prominent isoform, TG2, is associated with physiological processes such as extracellular matrix (ECM) stabilization and plays a crucial role in the pathogenesis of e.g. fibrotic diseases, cancer and celiac disease. Therefore, TG2 represents a pharmacological target of increasing relevance. The glycosaminoglycans (GAG) heparin (HE) and heparan sulfate (HS) constitute high-affinity interaction partners of TG2 in the ECM. Chemically modified GAG are promising molecules for pharmacological applications as their composition and chemical functionalization may be used to tackle the function of ECM molecular systems, which has been recently described for hyaluronan (HA) and chondroitin sulfate (CS). Herein, we investigate the recognition of GAG derivatives by TG2 using an enzyme-crosslinking activity assay in combination with in silico molecular modeling and docking techniques. The study reveals that GAG represent potent inhibitors of TG2 crosslinking activity and offers atom-detailed mechanistic insights.

Collagen/hyaluronan based hydrogels releasing sulfated hyaluronan improve dermal wound healing in diabetic mice via reducing inflammatory macrophage activity.

Sustained inflammation associated with dysregulated macrophage activation prevents tissue formation and healing of chronic wounds. Control of inflammation and immune cell functions thus represents a promising approach in the development of advanced therapeutic strategies. Here we describe immunomodulatory hyaluronan/collagen (HA-AC/coll)-based hydrogels containing high-sulfated hyaluronan (sHA) as immunoregulatory component for the modulation of inflammatory macrophage activities in disturbed wound healing. Solute sHA downregulates inflammatory activities of bone marrow-derived and tissue-resident macrophages in vitro. This further affects macrophage-mediated pro-inflammatory activation of skin cells as shown in skin ex-vivo cultures. In a mouse model of acute skin inflammation, intradermal injection of sHA downregulates the inflammatory processes in the skin. This is associated with the promotion of an anti-inflammatory gene signature in skin macrophages indicating a shift of their activation profile. For in vivo translation, we designed HA-AC/coll hydrogels allowing delivery of sHA into wounds over a period of at least one week. Their immunoregulatory capacity was analyzed in a translational experimental approach in skin wounds of diabetic db/db mice, an established model for disturbed wound healing. The sHA-releasing hydrogels improved defective tissue repair with reduced inflammation, augmented pro-regenerative macrophage activation, increased vascularization, and accelerated new tissue formation and wound closure.

Sulfated hyaluronan derivatives reduce the proliferation rate of primary rat calvarial osteoblasts.

Glycosaminoglycans (GAG) and proteoglycans, which are components of the extracellular bone matrix, are also localized in and at the membrane of osteoblasts and in the pericellular matrix. Due to their interaction with several growth factors, water and cations these molecules play an important role in regulating proliferation and differentiation of osteoblasts and bone development. The aim of this study was to assess in vitro the effects of two chemically sulfated hyaluronan (HyaS) derivatives on the proliferation of rat calvarial osteoblasts and to compare with those of native hyaluronan (Hya) and natural sulfated GAG such as chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS) and heparan sulfate (HS). Moderately and highly sulfated HyaS derivatives caused a time-dependent reduction of osteoblast proliferation. The anti-proliferative effect of HyaS was accompanied by a cell cycle arrest in the G1 phase, but was not associated with cell death. Whereas non-sulfated high molecular weight (HMW)- and low molecular weight (LMW)-Hya as well as C4S, C6S, DS and HS showed no effect on the cell proliferation.