Tanmay Gharat1, Andrea C. Jimenez-Vergara2, Dany Munoz-Pinto2, Melissa Grunlan3, Mariah S. Hahn2
1 Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York
2 Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York
3 Department of Biomedical Engineering, Texas A&M University, College Station, Texas
Introduction: Tissue engineering is a promising alternative to conventional treatments such as micro fracture or grafting for the treatment of osteochondral defects. Traditional tissue engineering approaches to osteochondral regeneration typically involve a combination of two distinct scaffolds for cartilage and subchondral bone. However, such scaffolds are generally unable to recapitulate the native cartilage-to-bone transition needed for long-term mechanical stability1. Moreover, these scaffolds are also unable to achieve the gradual cell phenotypic transition from chondrocyte to osteoblasts found within the transition zone. Since mesenchymal stem cells (MSCs) have the unique ability to undergo differentiation under the influence of matrix-mediated cues, we propose hydrogel constructs incorporating gradient biochemical signals for stimulating spatially-graded MSC differentiation for osteochondral repair. Specifically, polyethylene glycol diacrylate (PEGDA)-based hydrogels were chosen for their modifiable properties and the ability to incorporate bioactive factors. Within this PEGDA base, we incorporated spatially decreasing levels of chondroitin sulfate (CSC) and transforming growth factor-beta1 (TGF-β1) for inducing decreasing levels of chondrogenesis with increasing construct depth2. Simultaneously, increasing concentrations of the polydimethylsiloxane (PDMS) (a polymer which has previously been shown to have osteoinductive capacity when combined with PEGDA) 3 and bone morphogenetic protein-2 (BMP-2) were used to achieve increasing MSC osteogenesis with increased construct depth. In this design, growth factors were tethered to the hydrogel network to enable prolonged signaling (allowing for reduced growth factor concentrations) and to enable localized delivery4, minimizing the potential for undesirable responses in surrounding tissues. To evaluate the capacity of these gradient hydrogels to drive spatially-specific osteochondral differentiation, MSCS derived from synovial fluid (SMSCs) 5 were encapsulated within: 1) a “high CSC, high TGF-β1” hydrogel [for cartilage], 2) a “high PDMS, high BMP-2” hydrogel [for bone], and 3) an “intermediate TGF-β1, PDMS and BMP-2” hydrogel [for the transition zone].
Methods: Synthesis of Macromers. PEGDA (3.4 kDa), PDMSstar-MA (2.5 kDa) and CSC-MA (51 kDa) were prepared as previously described3. Acrylate-derivatized TGF-β1, acrylate-derivatized BMP-2 and acrylate-derivatized cell adhesion ligand RGDS were synthesized per standard NHS-chemistry. Fabrication of constructs: Three hydrogel precursor solutions containing 10% wt. PEGDA, 1 mM acryloyl-RGDS and photoinitiator were prepared as follows: i) 5.0 mg/ml CSC-MA + 5.0 ng/ml acrylate-derivatized TGF-β1 (designated “Cartilage”) ii) 0.5 wt% PDMSstar-MA + 2.0 ng/ml acrylate-derivatized TGF-β1 + 20 ng/ml acrylate-derivatized BMP-2 (designated “Transition zone”) and iii) 2.0 wt% PDMSstar-MA + 50 ng/ml acrylate-derivatized BMP-2 (designated “Bone”) along with a PEGDA control containing no additives. Canine SMSCs were encapsulated within these hydrogels by 6 min exposure to 365 nm UV light. Construct Culture and Cell Characterization: Following 21 days of culture in media without supplements, the phenotype of encapsulated MSCs was analyzed using Western blotting.
Results and Discussion: SMSC protein-level expression of various cartilage and bone markers was analyzed in each hydrogel formulation following 21 days of culture (Figure 1). The ‘Cartilage' formulation showed a 1.2-fold and 2.2-fold increase for sox9 and collagen II (Col-2), respectively, relative to the PEGDA control. Furthermore, the levels of sox9 and Col-2 in the “Cartilage” formulation were also 1.5-fold and 2.4-fold higher than in the “Bone” hydrogels. These data indicate increased chondrogenesis within the “Cartilage” formulation. In contrast, the levels of osteogenic markers collagen I (Col-1) and alkaline phosphatase (TNAP) were 3.3-fold and 1.6-fold greater in the “Bone” formulation than in the PEGDA control hydrogels. These markers were also both approximately 2-fold higher in the “Bone” constructs relative to the “Cartilage” hydrogels, indicating increase osteogenesis in the “Bone” formulation. In the “Transition zone” hydrogels, levels of sox9 and Col-2 were intermediate between those observed in the “Cartilage” and “Bone” formulations. Furthermore, levels of TNAP, Col-I, and osterix were similar to or increased relative to the “Bone” formulation, suggesting mixed “Bone”/”Cartilage” cell behavior in the “Transition zone” formulation.
Conclusions: Overall, the results indicate a gradual transition from chondrocyte-like to osteoblast-like phenotype within the developed gradient hydrogels, as evident from SMSC neo-matrix deposition. In the long-term, scaffolds formed with spatial gradients of chondrogenic and osteogenic mediators may prove beneficial for osteochondral regeneration.
Acknowledgments: The authors gratefully acknowledge funding from the NIH, NIBIB and AKC.
References: 1. Keeney, M., Tissue Eng. Part B 15 (1), 55-73 (2009), 2. Varghese S., Matrix Biology 27, 12-21 (2008) 3. Munoz-Pinto, D. J., Tissue Eng. Part A 18, 1710–1719 (2012), 4. Chen, C., Knee Surgery, Sport. Traumatol. Arthrosc. 19, 1597–1607 (2011). 5. De Bari, C., Arthritis & Rheumatism 44 (8), 1928-1942 (2001).