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Future of Soft Tissue Regeneration: 3D Printed PCLF Scaffolds, Progenitor Cell Proliferation and Differentiation, and Vascularization with Tissue Infiltration

Posted on:2017-01-09Degree:M.SType:Thesis
University:College of Medicine - Mayo ClinicCandidate:Wagner, Eric RichardFull Text:PDF
GTID:2444390005478343Subject:Biomedical engineering
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Introduction: Soft tissue injuries around joints are particularly difficult to treat. Particularly when intra-articular, surgical reconstruction is hampered by poor regenerative potential of the tissue. Tissue engineering has attempted to combine scaffold technology with biologic regeneration to overcome these difficulties. The aim of this thesis was to use 3D printing and CAD design software technology to create a novel scaffold from a polymer (PCLF) that possesses an excellent cytocompatibility, a slowly biodegrading nature, and an ability to easily modify its biomechanical characteristics, such as strength and flexibility. The scaffold is then customized and analyzed for its potential in ligament engineering (first aim) and cartilage regeneration (second aim), as well as revascularization and tissue ingrowth (third aim).;Materials and Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF), then we designed via CAD software and used 3D printing technology to create macro-porous scaffolds with channels traversing the body in 3 axes to allow cell-cell communication and nutrient flow. The first aim utilized clinical grade human adipose-tissue derived human mesenchymal stem cells (AMSCs) and anterior cruciate ligament (ACL) fibroblasts, while the second aim utilized chondrocytes and fibroblasts from rabbits. Seeding of the scaffolds was performed using a dynamic bioreactor, while the culture media were either 10% fetal bovine serum (FBS) or 5% human platelet lysate (PL), with the addition of growth factors for the differentiation assays. Cell attachment, growth, viability and differentiation were examined using metabolic assays and immunostaining. In the last aim, the PCLF scaffolds were cross-linked with PLGA microspheres containing growth factors (VEGF for vascularization, FGF-2 for fibrous tissue ingrowth, and BMP-2 for bone ingrowth), and were implanted into the subcutaneous tissues of rats for 6 and 12 weeks. The scaffolds then underwent histological staining to assess for adverse immune response, tissue ingrowth, and vessel and bone formation. MicroCT angiography was used to assess formation of a vascular and bony network in the body's of the scaffolds.;Results: In the first project, AMSCs seeded on PCLF scaffolds remained viable for multiple weeks, proliferating in the presence of platelet lysate (PL) (p<0.05), and infiltrating throughout the pores and channels. Cells had a low baseline expression of osteogenic and chondrogenic markers, but increased expression of total collagen when induced by FGF2 and PL (p<0.01). FGF2 and PL also significantly increased immunostaining for AMSC expression of Tenascin-C and Collagen I, compared to control AMSCs.;In the second project, chondrocytes seeded on the PCLF scaffolds remained viable for over 2 weeks, proliferating at higher rates in the presence of PL (p<0.05). The chondrogenic markers and total collagen contents increased at each time point (p<0.05), while the osteogenic marker did not. Immunostaining at 2 and 4 weeks for the expression of chondrogenic markers Collagen II and Sox-9 were significantly increased when compared to control human fibroblasts.;In the last project, at the 12 week time point, the scaffolds had tissue infiltrating into their pores without any signs of an adverse immune response against the material, including favorable macrophage phenotypic ratios. Histologic analysis and MicroCT angiography demonstrated the scaffolds seeded with microspheres containing VEGF had significantly higher vascular ingrowth and vessel penetration than the control scaffolds (p<0.01). The scaffolds with BMP-2 (in addition to VEGF) had high levels of mineral deposition throughout the scaffold. Augmenting the scaffolds with VEGF alone, or in combination with BMP-2 or FGF-2 led to marked collagen tissue infiltration throughout the pores, those with BMP demonstrated mineral deposition into the pores, while the control scaffolds had increased fatty infiltration.;Conclusion: We have developed a novel cyto- and bio-compatible PCLF scaffold using 3D printing to customize a scaffold containing large pores and channels traversing the scaffold in 3 different axes. The large pores, surface roughness, friendly micro-environment, and cell friendly synthetic polymer creates an environment which progenitor and mature mesenchymal cells are able to attach, proliferate and remain viable, while inducing differentiation upon the scaffolds. Additionally, the biologically favorable structural (biologically friendly polymer), geometric (large channels and pores, rough surfaces), and regenerative (growth factor loaded microsphere cross-linkability) characteristics of these PCLF scaffolds lead to marked vessel and collagen tissue network formation.
Keywords/Search Tags:Tissue, PCLF scaffolds, 3D printing, Collagen, Differentiation, Cell, Regeneration, VEGF
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