gms | German Medical Science

Objectives: Despite intensive research efforts, an adequate regeneration of articular cartilage defects still represents a major clinical challenge. Our overall aim, therefore, is the development of an innovative platform of reactive poly(ethylene glycol) or polyglycidol cross-linked hydrogels for guided chondrogenesis. Here, we demonstrate functionalization by coupling of chondromimetic peptides and chondrogenic growth factors, and subsequent chondrogenic differentiation of bone marrow-derived mesenchymal stem cells [BMSC] within these gels.

Methods: Thiolated hyaluronic acid [HA] was cross-linked either with poly(ethylene glycol) [PEG, 4-arm, 8-arm] or polyglycidol [PG] derivatives using Michael addition or thiol-ene click chemistry. Alternatively, pure PG gels were generated by UV-mediated cross-linking of thiolated PG. Various chondromimetic peptides at different densities were incorporated into the gels by coupling to either PEG or PG. In a separate approach, transforming growth factor-β1 (TGF-β1) was covalently bound within the gels at various doses (10 - 100 nM). Gels were seeded with 8x10⁵ BMSCs per 40 µl gel and cells were chondrogenically differentiated for 21 days. Extracellular matrix (ECM) components such as glycosaminoglycans (GAG) and various collagens were analyzed histologically and immunohistochemically. GAG and collagen content was quantified biochemically. Furthermore, chondrogenesis was investigated on the mRNA level using qRT-PCR.

Results and Conclusion: In all unmodified PEG or PG cross-linked HA hydrogels, chondrogenic differentiation was visualized with staining for GAG and collagen II, but also collagen I was observed. This was reflected in GAG and collagen quantification and gene expression analysis. Incorporation of peptides derived from ECM molecules, e.g. KLER from decorin, modulated cartilage matrix composition. Strong peptide effects were especially observed in pure PG gels exhibiting distinctly higher numbers of peptide binding sites, as compared to PEG-derived gels. Incorporating N-cadherin-mimetic peptides, e.g. HAVDI, into pure PG gels led to clearly increased GAG content and alteration of collagen type ratio, as compared to scrambled peptides or unmodified gels. Covalent binding of TGF-β1 (100 nM) within the hydrogels led to strong chondrogenesis after 10 and 21 days, e.g., with distinctly increased absolute GAG and GAG/DNA content, as compared to constructs receiving equal amounts of TGF-β1 mixed within the gels or constructs receiving TGF-β1 (10 ng/ml) three times per week with each medium change.

These results confirm the suitability of the developed hydrogels for cartilage engineering and for analysis of peptide and growth factor effects on chondrogenesis. The strong chondrogenic effects of bound TGF-β1, i.e., without the necessity for a continuous application, render the gels especially attractive. In ongoing work, a combined coupling of biomimetic peptides and growth factors is explored to further improve the clinical potential of this material.