Scavenging of Dickkopf-1 by macromer-based biomaterials covalently decorated with sulfated hyaluronan displays pro-osteogenic effects.

Dickkopf-1 (DKK1), a Wnt inhibitor secreted by bone marrow stromal cells (MSC), is known to play an important role in long-term non-union bone fracture defects and glucocorticoid induced osteoporosis. Mitigating its effects in early bone defects could improve osteogenesis and bone defect healing. Here, we applied a biomaterial strategy to deplete a defect environment from DKK1 by scavenging the protein via a macromer-based biomaterial covalently decorated with sulfated hyaluronan (sHA3). The material consisted of cross-copolymerized three-armed macromers with a small anchor molecule. Using the glycidyl anchor, polyetheramine (ED900) could be grafted to the material to which sHA3 was efficiently coupled in a separate step. For thorough investigation of material modification, flat material surfaces were generated by fabricating them on glass discs. The binding capability of sHA3 for DKK1 was demonstrated in this study by surface plasmon resonance measurements. Furthermore, the surfaces demonstrated the ability to scavenge and inactivate pathologic amounts of DKK1 from complex media. In a combinatory approach with Wnt3a, we were able to demonstrate that DKK1 is the preferred binding partner of our sHA3-functionalized surfaces. We validated our findings in a complex in vitro setting of differentiating SaOS-2 cells and primary hMSC. Here, endogenous DKK-1 was scavenged resulting in increased osteogenic differentiation indicating that this is a consistent biological effect irrespective of the model system used. Our study provides insights in the mechanisms and efficiency of sHA3 surface functionalization for DKK1 scavenging, which may be used in a clinical context in the future.

Chondroitin sulfate and sulfated hyaluronan-containing collagen coatings of titanium implants influence peri-implant bone formation in a minipig model.

An improved osseous integration of dental implants in patients with lower bone quality is of particular interest. The aim of this study was to evaluate the effect of artificial extracellular matrix implant coatings on early bone formation. The coatings contained collagen (coll) in conjunction with either chondroitin sulfate (CS) or sulfated hyaluronan (sHya). Thirty-six screw-type, grit-blasted, and acid-etched titanium implants were inserted in the mandible of 6 minipigs. Three surface states were tested: (1) uncoated control (2) coll/CS (3) coll/sHya. After healing periods of 4 and 8 weeks, bone implant contact (BIC), bone volume density (BVD) as well as osteoid related parameters were measured. After 4 weeks, control implants showed a BIC of 44% which was comparable to coll/CS coated implants (48%) and significantly higher compared to coll/sHya coatings (37%, p = 0.012). This difference leveled out after 8 weeks. No significant differences could be detected for BVD values after 4 weeks and all surfaces showed reduced BVD values after 8 weeks. However, at that time, BVD around both, coll/CS (30%, p = 0.029), and coll/sHya (32%, p = 0.015), coatings was significantly higher compared to controls (22%). The osteoid implant contact (OIC) showed no significant differences after 4 weeks. After 8 weeks OIC for controls was comparable to coll/CS, the latter being significantly higher compared to coll/sHya (0.9% vs. 0.4%, p = 0.012). There were no significant differences in osteoid volume density. In summary, implant surface coatings by the chosen organic components of the extracellular matrix showed a certain potential to influence osseointegration in vivo.

Artificial extracellular matrices of collagen and sulphated hyaluronan enhance the differentiation of human mesenchymal stem cells in the presence of dexamethasone.

In this study we investigated the potential of artificial extracellular matrix (aECM) coatings containing collagen II and two types of glycosaminoglycan (GAGs) with different degrees of sulphation to promote human bone formation in biomedical applications. To this end their impact on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assessed. The cell proliferation was found to be significantly retarded in the first 14 days of culture on surfaces coated with collagen II and GAGs (coll-II/GAG) as compared to tissue culture polystyrol (TCPS) and those coated with collagen II. At later time points it only tended to be retarded on coll-II/sHya3.1. Heat-inactivation of the serum significantly reduced cell numbers on collagen II and coll-II/sHya3.1. Alkaline phosphatase (ALP) activity and calcium deposition, on the other hand, were higher for coatings containing sHya3.1 and were not significantly changed by heat-inactivation of the serum. Expression levels of the bone matrix proteins bone sialoprotein (BSP-II) and osteopontin (OP) were also increased on aECM coatings as compared to TCPS, which further validated the differentiation of hMSCs towards the osteogenic lineage. These observations reveal that aECM coatings, in particular those containing sHya3.1, are suitable to promote the osteogenic differentiation of hMSCs.

Poly(L-lactide-co-glycolide) scaffolds coated with collagen and glycosaminoglycans: impact on proliferation and osteogenic differentiation of human mesenchymal stem cells.

In this study, we analyzed poly(L-lactide-co-glycolide) (PLGA) scaffolds modified with artificial extracellular matrices (aECM) consisting of collagen type I, chondroitin sulphate, and sulphated hyaluronan (sHya). We investigated the effect of these aECM coatings on proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSC) in vitro. We found that scaffolds were homogeneously coated, and cross-linking of aECM did not significantly influence the amount of collagen immobilized. Cell proliferation was significantly increased on cross-linked surfaces in expansion medium (EM), but was retarded on cross-linked and non-cross-linked collagen/sHya coatings. The alkaline phosphatase activity was increased on sHya-containing coatings in EM even without the presence of differentiation supplements, but was six to ten times higher in differentiation medium (DM) and comparable for cross-linked and non-cross-linked collagen/sHya. The highest amount of calcium phosphate mineral was deposited on day 28 on cross-linked collagen/sHya. Therefore, coatings of PLGA scaffolds with collagen/sHya promoted the osteogenic differentiation of hMSCs in vitro and might be an interesting candidate for the modification of PLGA for bone reconstruction in vivo.

Sulfated hyaluronan/collagen I matrices enhance the osteogenic differentiation of human mesenchymal stromal cells in vitro even in the absence of dexamethasone.

Glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix (ECM) involved in different steps of the regulation of cellular differentiation. In this study artificial extracellular matrices (aECM) consisting of collagen (Col) I and different GAG derivatives were used as a substrate for human mesenchymal stromal cells (hMSC) to study osteogenic differentiation in vitro. hMSC were cultured on aECM containing col and hyaluronan sulfates (HyaS) with increasing degrees of sulfation (DS(S)) and were compared with aECM containing col and the natural GAG hyaluronan or chondroitin 4-sulfate. hMSC were analyzed for osteogenic differentiation markers such as calcium phosphate deposition, tissue non-specific alkaline phosphatase (TNAP) and expression of runt-related transcription factor 2 (runx2), osteocalcin (ocn) and bone sialoprotein II (bspII). Compared with aECM containing Col and natural GAG all Col/HyaS-containing aECM induced an increase in calcium phosphate deposition, TNAP activity and tnap expression. These effects were also seen in the absence of dexamethasone (an established osteogenic supplement). The expression of runx2 and ocn was not altered and the expression of bspII was diminished on the col/HyaS-containing aECM. The impact of the Col/HyaS-containing aECM on hMSC differentiation was independent of the DS(S) of the HyaS derivatives, indicating the importance of the primary (C-6) hydroxyl group of N-acetylglucosamine. These results suggest that Col/HyaS-containing aECM are able to stimulate hMSC to undergo osteogenic differentiation even in the absence of dexamethasone, which makes these matrices an interesting tool for hMSC-based tissue engineering applications and biomaterial functionalizations to enhance bone formation.

Sulfated hyaluronan and chondroitin sulfate derivatives interact differently with human transforming growth factor-ß1 (TGF-ß1).

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-ß1 (TGF-ß1) 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-ß1, 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.