Vera Hintze

Modifications of hyaluronan influence the interaction with human bone morphogenetic protein-4 (hBMP-4).

In this study, we have demonstrated that the modification of hyaluronan (hyaluronic acid; Hya) with sulfate groups led to different binding affinities for recombinant human bone morphogenetic protein-4 (rhBMP-4). The high-sulfated sHya2.8 (average degree of sulfation (D.S.) 2.8) exhibited the tightest interaction with rhBMP-4, followed by the low-sulfated sHya1.0, as determined with surface plasmon resonance (SPR), ELISA, and competition ELISA. Unmodified Hya, chondroitin-sulfate (CS), and heparan sulfate (HS) showed significantly less binding affinity. SPR data could be fitted to an A + B = AB Langmuir model and binding constants were evaluated ranging from 13 pM to 5.45 microM. The interaction characteristics of the differentially sulfated Hyas are promising for the incorporation of these modified polysaccharides in bioengineered coatings of biomaterials for medical applications.

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-β1 (TGF-β1)

This study demonstrates that the modification of hyaluronan (hyaluronic acid; Hya) and chondroitin sulfate (CS) with sulfate groups leads to different binding affinities for recombinant human transforming growth factor-β1 (TGF-β1) for comparable average degrees of sulfation (DS). In general, Hya derivates showed higher binding strength than CS derivatives. In either case, a higher degree of sulfation leads to a stronger interaction. The high-sulfated hyaluronan sHya3 (average DS≈3) exhibited the tightest interaction with TGF-β1, as determined by surface plasmon resonance and enzyme-linked immunosorbent assay. The binding strength was significantly weakened by carboxymethylation. Unmodified Hya and low-sulfated, native CS showed weak or no binding affinity. The interaction characteristics of the different sulfated glycosaminoglycans are promising for incorporation into bioengineered coatings of biomaterials to modulate growth factor binding in medical applications.

Artificial extracellular matrices composed of collagen I and sulfated hyaluronan with adsorbed transforming growth factor β1 promote collagen synthesis of human mesenchymal stromal cells.

Sulfated glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix and are involved in the regulation of adhesion, proliferation and differentiation of cells. The effects of GAG are mediated in general by their interactions with cations and water, and in particular by their binding to growth factors. The aim of this study was to generate artificial extracellular matrices (aECM) containing collagen I and hyaluronan sulfate (HyaS), which are capable of adsorbing and releasing transforming growth factor β1 (TGF-β1), and to promote collagen synthesis of cultured human mesenchymal stromal cells (hMSC). For the preparation of aECM, monosulfated Hya (HyaS1) or trisulfated Hya (HyaS3) were used; the natural chondroitin-4-sulfate was used as a control. As applied for the in vitro experiments, the resulting matrices were composed of 93-98% collagen I and 2-7% GAG derivative. Adsorption of TGF-β1 to the aECM and release from the aECM was dependent on the degree of sulfation of hyaluronan. Collagen synthesis of hMSC was promoted only by aECM with adsorbed TGF-β1; the bare aECM had a slightly inhibitory effect on collagen synthesis. The promoting effect did not correlate either to the amount of adsorbed TGF-β1 nor to the release of TGF-β1, indicating that the correct presentation of TGF-β1 to the cells might be critical. The results indicate that sulfated hyaluronan-containing aECM have the potential to control both the adsorption and release of TGF-β1, and thereby promote collagen synthesis of hMSC. Thus, these aECM might be a useful tool for different tissue-engineering applications to enhance bone formation when used for biomaterial coating.

Biological functionalization of dental implants with collagen and glycosaminoglycans—A comparative study

Biological implant surface coatings are an emerging technology to increase bone formation. Such an approach is of special interest in anatomical regions like the maxilla. In the present study, we hypothesized that the coating of titanium implants with components of the organic extracellular matrix increases bone formation and implant stability compared to an uncoated reference. The implants were coated using collagen-I with either two different concentrations of chondroitin sulfate (CS) or two differentially sulfated hyaluronans. Implant coatings were characterized biochemically and with atomic force microscopy. Histomorphometry was used to assess bone-implant contact (BIC) and bone-volume density (BVD) after 4 and 8 weeks of submerged healing in the maxilla of 20 minipigs. Further, implant stability was measured by resonance frequency analysis (RFA). Implants containing the lower CS concentration had significantly more BIC, compared to the uncoated reference at both times of interest. No significant increase was measured from week 4 to 8. Differences in BVD and RFA were statistically not significant. A higher concentration of CS and the application of sulfated hyaluronans showed no comparable increase in BIC. This study demonstrates a positive effect of a specific collagen-glycosaminoglycan combination on early bone formation in vivo.

Artificial extracellular matrices composed of collagen I and high sulfated hyaluronan modulate monocyte to macrophage differentiation under conditions of sterile inflammation.

Integration of biomaterials into tissues is often disturbed by unopposed activation of macrophages. Immediately after implantation, monocytes are attracted from peripheral blood to the implantation site where they differentiate into macrophages. Inflammatory signals from the sterile tissue injury around the implanted biomaterial mediate the differentiation of monocytes into inflammatory M1 macrophages (M1) via autocrine and paracrine mechanisms. Suppression of sustained M1 differentiation is thought to be crucial to improve implant healing. Here, we explore whether artificial extracellular matrix (aECM) composed of collagen I and hyaluronan (HA) or sulfated HA-derivatives modulate this monocyte differentiation. We mimicked conditions of sterile tissue injury in vitro using a specific cytokine cocktail containing MCP-1, IL-6 and IFNγ, which induced in monocytes a phenotype similar to M1 macrophages (high expression of CD71, HLA-DR but no CD163 and release of high amounts of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12 and TNFα). In the presence of aECMs containing high sulfated HA this monocyte to M1 differentiation was disturbed. Specifically, pro-inflammatory functions were impaired as shown by reduced secretion of IL-1β, IL-8, IL-12 and TNFα. Instead, release of the immunregulatory cytokine IL-10 and expression of CD163, both markers specific for anti-inflammatory M2 macrophages (M2), were induced. We conclude that aECMs composed of collagen I and high sulfated HA possess immunomodulating capacities and skew monocyte to macrophage differentiation induced by pro-inflammatory signals of sterile injury toward M2 polarization suggesting them as an effective coating for biomaterials to improve their integration.

Synergistic effect of defined artificial extracellular matrices and pulsed electric fields on osteogenic differentiation of human MSCs

In vivo, bone formation is a complex, tightly regulated process, influenced by multiple biochemical and physical factors. To develop a vital bone tissue engineering construct, all of these individual components have to be considered and integrated to gain an in vivo-like stimulation of target cells. The purpose of the present studies was to investigate the synergistic role of defined biochemical and physical microenvironments with respect to osteogenic differentiation of human mesenchymal stem cells (MSCs). Biochemical microenvironments have been designed using artificial extracellular matrices (aECMs), containing collagen I (coll) and glycosaminoglycans (GAGs) like chondroitin sulfate (CS), or a high-sulfated hyaluronan derivative (sHya), formulated as coatings on three-dimensional poly(caprolactone-co-lactide) (PCL) scaffolds. As part of the physical microenvironment, cells were exposed to pulsed electric fields via transformer-like coupling (TC). Results showed that aECM containing sHya enhanced osteogenic differentiation represented by increases in ALP activity and gene-expression (RT-qPCR) of several bone-related proteins (RUNX-2, ALP, OPN). Electric field stimulation alone did not influence cell proliferation, but osteogenic differentiation was enhanced if osteogenic supplements were provided, showing synergistic effects by the combination of sHya and electric fields. These results will improve the understanding of bone regeneration processes and support the development of effective tissue engineered bone constructs.

The effect of the degree of sulfation of glycosaminoglycans on osteoclast function and signaling pathways

To meet the growing need for bone replacement of our aging population, development of new adaptive biomaterials is essential. Collagen and glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major components of the extracellular matrix (ECM) in bone. We manufactured native and sulfate-modified GAG matrices, evaluated how these components modulate different functions of osteoclasts, the cells that resorb bone, and analyzed the underlying mechanisms. GAGs were tested for their effects on osteoclast adhesion, viability, differentiation, morphology, and resorption as well as proteome alterations using murine RAW264.7 cells and primary human osteoclasts. Native and sulfated GAGs were stable and largely non-cytotoxic. Sulfation of GAGs led to a significant inhibition of osteoclast differentiation and resorption, which was largely dependent on the degree of sulfation of GAGs rather than the monosaccharide composition. Sulfation significantly reduced resorptive function by 14% (CS) and 43% (HA). Highly sulfated GAGs dose-dependently suppressed osteoclast differentiation, osteoclast-specific expression of TRAP, cathepsin K, SWAP-70, and OSCAR by 63-95%, and inhibited proteins involved in cytoskeletal rearrangement. In conclusion, highly sulfated GAGs significantly inhibit various functions of bone-resorbing osteoclasts. Whether these properties locally contribute to improved fracture or bone defect healing needs to be validated in vivo.

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.

Sulfated Glycosaminoglycans Exploit the Conformational Plasticity of Bone Morphogenetic Protein-2 (BMP-2) and Alter the Interaction Profile with Its Receptor

Sulfated glycosaminoglycans (GAGs) can direct cellular processes by interacting with proteins of the extracellular matrix (ECM). In this study we characterize the interaction profiles of chemically sulfated hyaluronan (HA) and chondroitin sulfate (CS) derivatives with bone morphogenetic protein-2 (BMP-2) and investigate their relevance for complex formation with the receptor BMPR-IA. These goals were addressed by surface plasmon resonance (SPR) and ELISA in combination with molecular modeling and dynamics simulation. We found not only the interaction of BMP-2 with GAGs to be dependent on the type and sulfation of GAGs but also BMP-2/GAG/BMPR-IA complex formation. The conformational plasticity of the BMP-2 N-termini plays a key role in the structural and thermodynamic characteristics of the BMP-2/GAG/BMPR-IA system. Hence we propose a model that provides direct insights into the importance of the structural and dynamical properties of the BMP-2/BMPR-IA system for its regulation by sulfated GAGs, in which structural asymmetry plays a key role.

Functionalization of biomaterial surfaces using artificial extracellular matrices

Construction of biomaterials with the ability to guide cell function is a topic of high interest in biomaterial development. One approach is using components native to the ECM of the target tissue to generate in vitro a microenvironment that can also elicit specific responses in cells and tissues—an artificial ECM (aECM). The focus is on collagen as the basic material, which can be modified using a number of different glycoproteins, proteoglycans and glycosaminoglycans. Preparation, immobilization and the biochemical characteristics of such aECM are discussed, as well as the in vitro and in vivo response of cells and tissues, illustrating the potential of such matrices to direct cell fate.