Follistatin Effects in Migration, Vascularization, and Osteogenesis in vitro and Bone Repair in vivo

The use of biomaterials and signaling molecules to induce bone formation is a promising approach in the field of bone tissue engineering. Follistatin (FST) is a glycoprotein able to bind irreversibly to activin A, a protein that has been reported to inhibit bone formation. We investigated the effect of FST in critical processes for bone repair, such as cell recruitment, osteogenesis and vascularization, and ultimately its use for bone tissue engineering. In vitro, FST promoted mesenchymal stem cell (MSC) and endothelial cell (EC) migration as well as essential steps in the formation and expansion of the vasculature such as EC tube-formation and sprouting. FST did not enhance osteogenic differentiation of MSCs, but increased committed osteoblast mineralization. In vivo, FST was loaded in an in situ gelling formulation made by alginate and recombinant collagen-based peptide microspheres and implanted in a rat calvarial defect model. Two FST variants (FST288 and FST315) with major differences in their affinity to cell-surface proteoglycans, which may influence their effect upon in vivo bone repair, were tested. In vitro, most of the loaded FST315 was released over 4 weeks, contrary to FST288, which was mostly retained in the biomaterial. However, none of the FST variants improved in vivo bone healing compared to control. These results demonstrate that FST enhances crucial processes needed for bone repair. Further studies need to investigate the optimal FST carrier for bone regeneration

Sphere caging by a random ÿbre network

Abstract We analyse the remarkable e ciency of a random distribution of rigid thin rods (with diameter ) to 'cage' a test sphere (with diameter D ) by purely geometric hindrance due to rod-sphere contacts. The average number of random contacts which traps a sphere in three dimensions corresponds to a volume fraction c = 7(D= ) 2 of very long rods or ÿbres. Some implications for conÿnements and dynamics of (colloidal) particles in ÿbre structures are discussed

Injectable BMP-2 delivery system based on collagen-derived microspheres and alginate induced bone formation in a time-and dose-dependent manner

\u3cp\u3eThe aim of the current study was to reduce the clinically used supra-physiological dose of bone morphogenetic protein-2 (BMP-2) (usually 1.5 mg/mL), which carries the risk of adverse events, by using a more effective release system. A slow release system, based on an injectable hydrogel composed of BMP-2-loaded recombinant collagen-based microspheres and alginate, was previously developed. Time- and dose-dependent subcutaneous ectopic bone formation within this system and bone regeneration capacity in a calvarial defect model were investigated. BMP-2 doses of 10 µg, 3 µg and 1 µg per implant (50 µg/mL, 15 µg/mL and 5 µg/mL, respectively) successfully induced ectopic bone formation subcutaneously in rats in a time- and dose-dependent manner, as shown by micro-computed tomography (µCT) and histology. In addition, the spatio-temporal control of BMP-2 retention was shown for 4 weeks in vivo by imaging of fluorescently-labelled BMP-2. In the subcritical calvarial defect model, µCT revealed a higher bone volume for the 2 µg of BMP-2 per implant condition (50 µg/mL) as compared to the lower dose used (0.2 µg per implant, 5 µg/mL). Complete defect bridging was obtained with 50 µg/mL BMP-2 after 8 weeks. The BMP-2 concentration of 5 µg/mL was not sufficient to heal a calvarial defect faster than the empty defect or biomaterial control without BMP-2. Overall, this injectable BMP-2 delivery system showed promising results with 50 µg/mL BMP-2 in both the ectopic and calvarial rat defect models, underling the potential of this composite hydrogel for bone regeneration therapies.\u3c/p\u3

Supramolecular modification of a sequence-controlled collagen-mimicking polymer

\u3cp\u3eStructurally and functionally well-defined recombinant proteins are an interesting class of sequence-controlled macromolecules to which different crosslinking chemistries can be applied to tune their biological properties. Herein, we take advantage of a 571-residue recombinant peptide based on human collagen type I (RCPhC1), which we functionalized with supramolecular 4-fold hydrogen bonding ureido-pyrimidinone (UPy) moieties. By grafting supramolecular UPy moieties onto the backbone of RCPhC1 (UPy-RCPhC1), increased control over the polymer structure, assembly, gelation, and mechanical properties was achieved. In addition, by increasing the degree of UPy functionalization on RCPhC1, cardiomyocyte progenitor cells were cultured on soft (?26 kPa) versus stiff (?68-190 kPa) UPy-RCPhC1 hydrogels. Interestingly, increased stress fiber formation, focal adhesions, and proliferation were observed on stiffer compared to softer substrates, owing to the formation of stronger cell-material interactions. In conclusion, a bioinspired hydrogel material was designed by a combination of two well-known natural components, i.e., a protein as sequence-controlled polymer and UPy units inspired on nucleobases.\u3c/p\u3

Development of recombinant collagen-peptide-based vehicles for delivery of adipose-derived stromal cells

Stem cell therapy is a promising approach for repair, remodeling and even regenerate tissue of otherwise irreparable damage, such as after myocardial infarction (aMI). A severe limitation of cardiac stem cell therapy is the generally poor retention of administered cells in the target tissue. In tissue repair the main mode of action of adipose tissue-derived stem cells (ADSC) is the production of various growth factors, cytokines, anti-inflammatory and anti-apoptotic factors that together augment repair, remodeling, and regeneration. In this experiment, we used recombinant collagen peptide (RCP) with additional integrin-binding motives and different crosslinkers. Formulated as 50-100 mu m microspheres with bound ADSC, we hypothesized that this would improve ADSC retention and function. Crosslinking was performed with chemical crosslinkers (EDC and HMDIC) at high and low concentrations or by thermal treatment (DHT). ADSC adhesion, proliferation, apoptosis/necrosis, and gene expressions in two-dimensional and three-dimensional were analyzed. In addition, the effect of ADSC conditioned medium (ADSC-CM) on proapoptotic/sprouting HUVEC was examined. Our results show that all materials support cell adhesion in short time point, however, EDC-High crosslinker induced ADSC apoptosis/necrosis. Gene expression results revealed lower expression of proinflammatory genes in chemical crosslinked materials, despite EDC-High the proinflammatory genes expressions were similar or higher than TCPS. In addition, cultured ADSC on DHT crosslinked RCP showed a proinflammatory phenotype compared to TCPS. Sprouting assay results confirmed the protective effect of ADSC-CM derived from TCPS and HMDIC-High crosslinked RCP proapoptotic HUVEC. We conclude that ADSC adhere to the materials and maintain their therapeutic profile. (c) 2015 Wiley Periodicals, Inc

Collagen I derived recombinant protein microspheres as novel delivery vehicles for bone morphogenetic protein-2

\u3cp\u3eBone morphogenetic protein-2 (BMP-2) is a powerful osteoinductive protein; however, there is a need for the development of a safe and efficient BMP-2 release system for bone regeneration therapies. Recombinant extracellular matrix proteins are promising next generation biomaterials since the proteins are well-defined, reproducible and can be tailored for specific applications. In this study, we have developed a novel and versatile BMP-2 delivery system using microspheres from a recombinant protein based on human collagen I (RCP). In general, a two-phase release pattern was observed while the majority of BMP-2 was retained in the microspheres for at least two weeks. Among different parameters studied, the crosslinking and the size of the RCP microspheres changed the in vitro BMP-2 release kinetics significantly. Increasing the chemical crosslinking (hexamethylene diisocyanide) degree decreased the amount of initial burst release (24 h) from 23% to 17%. Crosslinking by dehydrothermal treatment further decreased the burst release to 11%. Interestingly, the 50 and 72 μm-sized spheres showed a significant decrease in the burst release compared to 207-μm sized spheres. Very importantly, using a reporter cell line, the released BMP-2 was shown to be bioactive. SPR data showed that N-terminal sequence of BMP-2 was important for the binding and retention of BMP-2 and suggested the presence of a specific binding epitope on RCP (K \u3csub\u3eD\u3c/sub\u3e: 1.2 nM). This study demonstrated that the presented RCP microspheres are promising versatile BMP-2 delivery vehicles. \u3c/p\u3