The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Following injuries and surgical repair, the enthesis is often not reestablished and so far, traditionally used tissue substitutes have lacked to reproduce the complexity of the native tissue. In this work, we hypothesised that a collagen-based three-layer scaffold that mimic the composition of the enthesis, in combination with bioactive molecules, will enhance the functional regeneration of the enthesis. A three-layer sponge composed of a tendon-like layer (collagen I), a cartilage-like layer (collagen II) and a bone-like layer (collagen I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold porosity and structural continuity at the interfaces were assessed through SEM analysis. Bone-marrow derived stem cells (BMSCs) were seeded by syringe vacuum assisted technique on the scaffold. Scaffolds were cultured in basal media for 3 days before switching to differentiation media (chondrogenic, tenogenic and osteogenic). BMSCs metabolic activity, proliferation and viability were assessed by alamarBlue, PicoGreen and Live/Dead assays. At D21 the scaffolds were fixed, cryosectioned and Alizarin Red and Alcian Blue stainings were performed in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I and tenascin in the scaffolds was evaluated by immunofluorescence staining at D21 in order to assess tenogenic differentiation of BMSCs. Subsequently, the cartilage-like layer was functionalized with IGF-1, seeded with BMSCs and cultured in basal media up to D21. Structural continuity at the interfaces of the scaffolds was confirmed by SEM and scaffold porosity was assessed as >98%. The scaffolds supported cell proliferation and infiltration homogeneously throughout all the layers up to D21. Osteogenic differentiation of BMSC selectively in the bone-like layer was confirmed by Alizarin red staining in scaffolds cultured in basal and osteogenic media. Alcian blue staining revealed the presence of proteoglycans selectively in the cartilage-like layer in scaffolds cultured in chondrogenic media but not in basal media. Increased expression of the tenogenic markers collagen I and tenascin were observed in the tendon-like layer of scaffolds cultured in tenogenic but not in basal media for 21 days. The presence of IGF-1 increased osteogenic and chondrogenic differentiation of BMSCs, whereas no difference was observed for tenogenic differentiation. In conclusion, a 3-layer collagen sponge was successfully fabricated with distinct but integrated layers; the different collagen composition of the non-functionalized 3-layer sponge was able to regulate BMSC differentiation in a localized manner within the scaffold. The scaffold functionalization with IGF-1 accelerated chondrogenic and osteogenic BMSC differentiation. Overall, functionalization of the 3-layer scaffolds holds promising potential in enthesis regeneration.

The enthesis is a tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Given the avascularity of the tendon and the gradual change in tissue architecture and cell phenotype, the enthesis original tissue is often not re-established after chronic injuries, resulting in scar formation. Conservative treatments and surgical approaches are still far from a functional regeneration, whilst tissue engineering based scaffolds have recently showed great potential. In this work, we hypothesised that collagen-based scaffolds that mimic the basic architecture of the enthesis, will be able to spatially direct stem cell differentiation, providing an in vitro platform to study enthesis regeneration.

A three-layer sponge composed of a tendon-like layer (collagen type I), a fibrocartilage-like layer (collagen type II) and a bone-like layer (collagen type I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold pore size and structural continuity at the interfaces were assessed by SEM and μ-CT analysis. Bone-marrow derived stem cells (BMSCs) were seeded on the scaffold and cultured in basal and differentiation media (chondrogenic, tenogenic and osteogenic). At day 7 and 21 the scaffolds were stained with Alizarin Red and Alcian Blue; alkaline phosphatase activity (ALP) and calcium and glycosaminoglycans (GAGs) were quantified in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I, III, tenascin and decorin in the scaffolds was evaluated by immunofluorescence staining in order to evaluate tenogenic differentiation of BMSCs.

Scaffolds with three distinct but interconnected layers of collagen type I, collagen type II and collagen type I + hydroxyapatite were fabricated, with pore sizes in the range of 100–200 μm. Increased ALP and calcium levels were detected in a localised manner within the bone-like layer when scaffolds were cultured in basal medium (p<0.025 vs the other 2 layers). Similarly, proteoglycans were detected specifically in the fibrocartilage-like layer when scaffolds were cultured in the chondrogenic differentiation medium (p<0.03 vs the other 2 layers). Increased expression of tenogenic markers was observed in the tendon-like layer of scaffolds cultured in tenogenic media (p<0.045 vs the other 2 layers).

In conclusion, the different collagen composition of each layer was able to spatially direct BMSC differentiation in a localized manner within the scaffold. Ongoing work is evaluating the synergistic effect between growth factor functionalized within the fibrocartilage and tendon-like layers for improved BMSC differentiation. Overall, these scaffolds hold promising potential in developing novel and more efficient strategy towards enthesis regeneration.

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones (tendon, fibrocartilage, mineralized fibrocartilage and bone). After injury, the native structure is often not re-established and a mechanically weaker fibrovascular scar is formed. Traditionally used monotherapies have failed to be effective, posing the need for multi-cargo localized delivery vehicles. We hypothesize that multilayer collagen-based scaffolds can serve as delivery vehicles for specific bioactive molecules with tenogenic, chondrogenic and osteogenic potential to enhance the functional regeneration of the enthesis. Three-layer scaffolds composed by a tendon-like layer of collagen type I, a cartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite were fabricated by an iterative layering freeze-drying technique. The scaffolds were cross-linked with varying concentration of 4-arm polyethylene glycol (4s-PEG) and the biological and mechanical properties were assessed. Each layer was functionalized with platelet-derived growth factor, insulin growth factor, heparan sulfate or bone morphogenetic protein 7 and their tenogenic, chondrogenic and osteogenic potential on bone-marrow derived stem cells was investigated in vitro. Scaffolds cross-linked with 1 mM 4s-PEG showed 60% free amines reduction respect to non-cross-linked scaffolds, were stable in collagenase over 24 hours and had a compression modulus of 30 kPa. The bioactive molecules had a sustained release profile (approximately 50 ng/mL) over 5 days as a function of cross-linking. Preliminary in vitro studies confirmed the chondrogenic potential of heparin sulfate and insulin growth factor by the increase of proteoglycans.