Prevention of Bone Bridge Formation Using Transplantation of the Autogenous Mesenchymal Stem Cells to Physeal Defects: An Experimental Study in Rabbits

Physeal cartilage is known to have poor self-repair capacity after injury. Evaluation of the ability of cultured mesenchymal stem cells to repair damaged physis is the topic of current research. In 10 immature New Zealand white rabbits autogenous mesenchymal stem cells were transplanted into a iatrogenic physeal defect in a lateral portion of the distal growth plate of the right femur. The same defect without stem cells transplantation in the left femoral distal physis served as a control. In our study, we used our own technique of implantation of MSCs with a newly modified gel scaffold (New Composite Hyaluronate/Collagen Type I/Fibrin Scaffold). The rabbits were euthanized 4 months after transplantation. Bone length discrepancy and valgus deformity were measured from femoral radiographs. Healing of the defect was investigated histologically. The ability of mesenchymal stem cells to survive and promote cartilage healing in the physeal defect was assessed by immunofluorescence. Average difference in femur length measured from surgery to euthanasia (4 months) was 0.61 ± 0.19 cm after preventive transplantation of MSCs in the right femur, but only 0.11 ± 0.07 cm in the left femur. Average angular (valgus) deformity of the right femur with MSCs preventively transplanted to iatrogenically damaged distal femoral physis was 1.2 ± 0.72 °. Valgus deformity in the left femur was 5.4 ± 2.5 °. Prophylactic transplantation of autogenous mesenchymal stem cells to iatrogenically damaged distal growth plate of the rabbit femur prevented a bone bridge formation and resulted in healing of the physeal defect with hyaline cartilage. Immunofluorescence examination showed that the chondrocytes newly formed in growth zone are the result of implanted MSCs differentiation. Femur growth in traumatized physis was maintained even after transplantation of autogenous MSCs. As compared with the opposite femur (with physeal defect but without transplanted MSCs), the bone showed no significant shortening or valgus deformity (p = 0.018)

Non-Woven PGA/PVA Fibrous Mesh as an Appropriate Scaffold for Chondrocyte Proliferation

Non-woven textile mesh from polyglycolic acid (PGA) was found as a proper material for chondrocyte adhesion but worse for their proliferation. Neither hyaluronic acid nor chitosan nor polyvinyl alcohol (PVA) increased chondrocyte adhesion. However, chondrocyte proliferation suffered from acidic byproducts of PGA degradation. However, the addition of PVA and/or chitosan into a wet-laid non-woven textile mesh from PGA improved chondrocyte proliferation seeded in vitro on the PGA-based composite scaffold namely due to a diminished acidification of their microenvironment. This PVA/PGA composite mesh used in combination with a proper hydrogel minimized the negative effect of PGA degradation without dropping positive parameters of the PGA wet-laid non-woven textile mesh. In fact, presence of PVA and/or chitosan in the PGA-based wet-laid non-woven textile mesh even advanced the PGA-based wet-laid non-woven textile mesh for chondrocyte seeding and artificial cartilage production due to a positive effect of PVA in such a scaffold on chondrocyte proliferation

Healing and angiogenic properties of collagen/chitosan scaffolds enriched with hyperstable FGF2-STAB® protein: in vitro, ex ovo and in vivo comprehensive evaluation

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration

An investigation of the influence of cartilaginous tissue microstructure on its local mechanical properties

This action is realized by the project NEXLIZ - CZ.1.07/2.3.00/30.0038, which is co-financed by the European social fund and the state budget of the Czech republic

Synergistic effect of bovine platelet lysate and various polysaccharides on the biological properties of collagen-based scaffolds for tissue engineering : scaffold preparation, chemo-physical characterization, in vitro and ex ovo evaluation

Crosslinked 3D porous collagen-polysaccharide scaffolds, prepared by freeze-drying, were modified with bovine platelet lysate (BPL) and evaluated in terms of chemical, physical and biological properties. Natural antibacterial polysaccharides like chitosan, chitin/chitosan-glucan complex and calcium salt of oxidized cellulose (CaOC) incorporated in collagen scaffolds affected not only chemo-physical properties of the composite scaffolds but also improved their biological properties, especially when BPL was presented. Lipophilic BPL formed microspheres in porous scaffolds while reduced by half their swelling ratio. The resistance of collagen sponges to hydrolytic degradation in water depended strongly on chemical crosslinking varying from 60 min to more than one year. According to in-vitro tests, chemically crosslinked scaffolds exhibited a good cellular response, cell–matrix interactions, and biocompatibility of the material. The combination of collagen with natural polysaccharides confirmed a significant positive synergistic effect on cultivation of cells as determined by MTS assay and PicoGreen method, as well as on angiogenesis evaluated by ex ovo Chick Chorioallantoic Membrane (CAM) assay. Contrary, modification only by BLP of pure collagen scaffolds exhibited decreased biocompatibility in comparison to unmodified pure collagen scaffold. We propose that the newly developed crosslinked collagen sponges involving bioactive additives could be used as scaffold for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration

Platelet-functionalized three-dimensional poly-ε-caprolactone fibrous scaffold prepared using centrifugal spinning for delivery of growth factors

Michala Rampichová,1,2 Matej Buzgo,1 Andrea Míčková,1,2 Karolína Vocetková,2 Vera Sovková,2 Vera Lukášová,2 Eva Filová,2 Franco Rustichelli,2 Evžen Amler1,2 1Indoor Environmental Quality, University Center for Energy Efficient Buildings, Czech Technical University in Prague, Buštehrad, 2Laboratory of Tissue Engineering, Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic Abstract: Bone and cartilage are tissues of a three-dimensional (3D) nature. Therefore, scaffolds for their regeneration should support cell infiltration and growth in all 3 dimensions. To fulfill such a requirement, the materials should possess large, open pores. Centrifugal spinning is a simple method for producing 3D fibrous scaffolds with large and interconnected pores. However, the process of bone regeneration is rather complex and requires additional stimulation by active molecules. In the current study, we introduced a simple composite scaffold based on platelet adhesion to poly-ε-caprolactone 3D fibers. Platelets were used as a natural source of growth factors and cytokines active in the tissue repair process. By immobilization in the fibrous scaffolds, their bioavailability was prolonged. The biological evaluation of the proposed system in the MG-63 model showed improved metabolic activity, proliferation and alkaline phosphatase activity in comparison to nonfunctionalized fibrous scaffold. In addition, the response of cells was dose dependent with improved biocompatibility with increasing platelet concentration. The results demonstrated the suitability of the system for bone tissue. Keywords: centrifugal spinning, 3D scaffold, platelets, growth factors, cytokines, PC