Growth Factor-releasing Systems Research Articles

Breathing new life into tissue engineering: exploring cutting-edge vascularization strategies for skin substitutes.

Tissue-engineered skin substitutes (TESS) emerged as anew therapeutic option to improve skin transplantation. However, establishing an adequate and rapid vascularization in TESS is a critical factor for their clinical application and successful engraftment in patients. Therefore, several methods have been applied to improve the vascularization of skin substitutes including (i) modifying the structural and physicochemical properties of dermal scaffolds; (ii) activating biological scaffolds with growth factor-releasing systems or gene vectors; and (iii) developing prevascularized skin substitutes by loading scaffolds with capillary-forming cells. This review provides a detailed overview of the most recent and important developments in the vascularization strategies for skin substitutes. Onthe one hand, we present cell-based approaches using stem cells, microvascular fragments, adipose tissue derived stromal vascular fraction, endothelial cells derived from blood and skin as well as other pro-angiogenic stimulation methods. On the other hand, we discuss how distinct 3D bioprinting techniques and microfluidics, miRNA manipulation, cell sheet engineering and photosynthetic scaffolds like GelMA, can enhance skin vascularization for clinical applications. Finally, we summarize and discuss the challenges and prospects of the currently available vascularization techniques that may serve as a steppingstone to a mainstream application of skin tissue engineering.

Osteogenic differentiation system based on biopolymer nanoparticles for stem cells in simulated microgravity

An efficient long-term intracellular growth factor release system in simulated microgravity for osteogenic differentiation was prepared based on polylactic acid (PLA) and polyhydroxyalkanoate (PHA) nanoparticles (NPs) for loading of bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 7 (BMP7) (defined as sB2-PLA-NPs and sB7-PHA-NPs), respectively, associated with osteogenic differentiation of human adipose derived stem cells (hADSCs). On account of soybean lecithin (SL) as biosurfactants, sB2-PLA-NPs and sB7-PHA-NPs had a high encapsulation efficiency (>80%) of BMPs and uniform small size (<100 nm), and showed a different slow-release to provide BMP2 in early stage and BMP7 in late stages of osteogenic differentiation within 20 d, due to degradation rate of PLA and PHA in cells. After uptake into hADSCs, by comparison with single sB2-PLA-NPs or sB7-PHA-NPs, the Mixture NPs compound of sB2-PLA-NP and sB7-PHA-NP with a mass ratio of 1:1, can well-promote ALP activity, expression of OPN and upregulated related osteo-genes. Directed osteo-differentiation of mixture NPs was similar to result of sustained free-BMP2 and BMP7-supplying (sFree-B2&B7) in simulated microgravity, which demonstrated the reliability and stability of Mixture NPs as a long-term osteogenic differentiation system in space medicine and biology in future.

Gene transfection achieved by utilizing antibacterial calcium phosphate nanoparticles for enhanced regenerative therapy

Protamine-coated multi-shell calcium phosphate (CaP) was developed as a non-viral vector for tissue regeneration therapy. CaP nanoparticles loaded with different amounts of plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) were used to treat MC3T3E1 cells, and the yield of the released BMP-2 or IGF-1 was measured using ELISA 3 days later. Collagen scaffolds containing CaP nanoparticles were implanted into rat cranial bone defects, and BMP-2 and IGF-1 yields, bone formation, and bone mineral density enhancement were evaluated 28 days after gene transfer. The antibacterial effects of CaP nanoparticles against Streptococcus mutans and Aggregatibacter actinomycetemcomitans increased with an increase in the protamine dose, while they were lower for Staphylococcus aureus and Porphyromonas gingivalis. In the combination treatment with BMP-2 and IGF-1, the concentration ratio of BMP-2 and IGF-1 is an important factor affecting bone formation activity. The calcification activity and OCN mRNA of MC3T3E1 cells subjected to a BMP-2:IGF-1 concentration ratio of 1:4 was higher at 14 days. During gene transfection treatment, BMP-2 and IGF-1 were released simultaneously after gene transfer; the loaded dose of the plasmid DNA encoding IGF-1 did not impact the BMP-2 or IGF-1 yield or new bone formation ratio in vitro and in vivo. In conclusion, two growth factor-releasing systems were developed using an antibacterial gene transfer vector, and the relationship between the loaded plasmid DNA dose and resultant growth factor yield was determined in vitro and in vivo.

Controlled Co-delivery of Growth Factors through Layer-by-Layer Assembly of Core-Shell Nanofibers for Improving Bone Regeneration.

The regeneration of bone tissue is regulated by both osteogenic and angiogenic growth factors which are expressed in a coordinated cascade of events. The aim of this study was to create a dual growth factor-release system that allows for time-controlled release to facilitate bone regeneration. We fabricated core-shell SF/PCL/PVA nanofibrous mats using coaxial electrospinning and layer-by-layer (LBL) techniques, where bone morphogenetic protein 2 (BMP2) was incorporated into the core of the nanofibers and connective tissue growth factor (CTGF) was attached onto the surface. Our study confirmed the sustained release of BMP2 and a rapid release of CTGF. Both in vitro and in vivo experiments demonstrated improvements in bone tissue recovery with the dual-drug release system. In vivo studies showed improvement in bone regeneration by 43% compared with single BMP2 release systems. Time-controlled release enabled by the core-shell nanofiber assembly provides a promising strategy to facilitate bone healing.

Enzymatic crosslinked gelatin 3D scaffolds for bone tissue engineering

Bone tissue engineering is an emerging medical field that has been developed in recent years to address pathologies with limited ability of bones to regenerate. Here we report the fabrication and characterization of microbial transglutaminase crosslinked gelatin-based scaffolds designed for serving as both cell substrate and growth factor release system. In particular, morphological, biomechanical and biological features have been analyzed. The enzyme ratio applied during the fabrication of the scaffolds affects the swelling capacity and the mechanical properties of the final structure. The developed systems are not cytotoxic according to the biocompatibility tests. The biological performance of selected formulations was studied using L-929 fibroblasts, D1 MSC and MG63 osteoblasts. Moreover, scaffolds allowed efficient osteogenic differentiation and signaling of MSCs. MSC cultured on the scaffolds not only presented lower proliferative and stemness profile, but also increased expression of osteoblast-related genes (Col1a1, Runx2, Osx). Furthermore, the in vitro release kinetics of vascular endothelial growth factor (VEGF) and bone morphogenetic protein −2 (BMP-2) from the scaffolds were also investigated. The release of the growth factors produced from the scaffolds followed a first order kinetics. These results highlight that the scaffolds designed and developed in this work may be suitable candidates for bone tissue regeneration purposes.

3D Bioprinting: Biologically Inspired Smart Release System Based on 3D Bioprinted Perfused Scaffold for Vascularized Tissue Regeneration (Adv. Sci. 8/2016)

In the article 1600058, Lijie Grace Zhang and co‐workers develop a comprehensive design of engineered vascularized bone construct (top image), integrating biomimetic 3D printed fluid perfused microstructure with a biologically inspired smart growth factor release system (colored fibers). The design provides great potential for complex tissue/organ regeneration by delivering multiple growth factors (small spheres), in a highly coordinated manner based on appropriate time, location and biological signal.

Development of a dual growth factor loaded biodegradable hydrogel and its evaluation on osteoblast differentiation in vitro

ABSTRACTHydrogels with their tunable properties are attractive candidates for developing tissue engineering scaffolds for various applications (including bone and cartilage). The current work involved studying the synergistic effect of basic fibroblast growth factor (bFGF) and platelet derived growth factor BB (PDGF-BB) entrapped within injectable porous gels for bone regeneration applications. An in situ gelling system was developed using bacterial polysaccharides gellan and xanthan gum by temperature and ionic gelation with Ca+2. After the initial characterization of the hydrogels, a dual growth factor release system was developed wherein growth factors were encapsulated within chitosan nanoparticles embedded in the gels as well as directly within the gel. The hydrogel structure was characterized by SEM and TEM and in vitro growth factor release studies showed a slow release profile in PBS. Further, human fetal osteoblasts were entrapped within the hydrogel and a 21 day osteoblast differentiation study was conducted. An improvement in osteoblast total protein synthesis and collagen content was observed by day 21 compared to control gels without growth factors. Although further evaluation regarding mechanical properties and expression of osteogenic differentiation marker genes will be necessary, the present study suggests that injectable scaffolds can be used for the delivery of multiple growth promoting agents to support osteoblast differentiation.

Multimodal release of transforming growth factor-β1 and the BB isoform of platelet derived growth factor from PEGylated fibrin gels

We designed a growth factor release system to potentially stabilize neovascularization in the treatment of ischemic tissue. In this study, the release of PDGF-BB and TGF-β1 was controlled with distinct kinetics from injectable PEGylated fibrin gels. Growth factors can be loaded into PEGylated fibrin gels via 3 mechanisms: entrapment, conjugation through a homobifunctional amine reactive PEG linker, and physical affinity with the fibrin matrix. PDGF-BB was entrapped during thrombin-mediated crosslinking leading to a diffusion-controlled release over 2 days. TGF-β1 was both conjugated through the PEG linker and bound to the matrix via physical affinity, delaying the release rate of TGF-β1 up to 10 days. Further, the release rate was highly correlated to gel degradation rate indicating that TGF-β1 release is degradation-controlled. Therefore, by modulating the molar ratio of PEG to fibrinogen, we were able to control the release rate of TGF-β1 without altering the release kinetics of PDGF-BB. The bioactivity of loaded TGF-β1 was maintained upon release as evidenced by the inhibition of cell proliferation. This system could be expanded to incorporate growth factors loaded via 3 schemes with differing release rates from an injectable system allowing for a high degree of flexibility in other combinational drug delivery and tissue engineering systems.

Sequential BMP‐2/BMP‐7 delivery from polyester nanocapsules

The aim of this study was to develop a nanosized, controlled growth factor release system to incorporate into tissue engineering scaffolds and thus activate the cells seeded in the scaffold. Nanocapsules of poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were loaded with the bone morphogenetic proteins BMP-2 and BMP-7, respectively, and with bovine serum albumin (BSA), the model protein. BSA-loading efficiency and release kinetics were used to determine the most appropriate nanocapsule pair to achieve the delivery of growth factors in a sequential manner, as occurs in natural processes. BSA-encapsulation efficiency was highest when the polymer concentration used in the preparation of PLGA and PHBV nanocapsules was 10% (w/v) (84.75% and 16.72%, respectively). Release of BSA was faster from PLGA than it was from PHBV. Based on the encapsulation efficiency and release data, 10% PLGA and 10% PHBV nanocapsules were chosen to provide the early BMP-2 and later BMP-7 release, respectively. Simultaneous, sequential delivery and individual release of the BMPs were studied for 7, 14, and 21 days, using rat bone marrow mesenchymal stem cells. Individual BMP-2 release suppressed cell proliferation while providing higher alkaline phosphatase activity with respect to BMP-7. The sequential delivery of BMP-2 and BMP-7 provided slightly lower proliferation than did simultaneous delivery, but the highest alkaline phosphatase activity of all indicated a synergistic effect on the osteogenic differentiation of mesenchymal stem cells caused by the use of the two growth factors in a sequential fashion.

Angiogenic growth factor release system for in vivo tissue engineering: a trial of bone marrow transplantation into ischemic myocardium

For successful in vivo tissue engineering, a growth factor release system will be useful. We adopted autologous bone marrow transplantation as an angiogenic growth factor release system. Bone marrow transplanted into a synthetic vascular prosthesis produced continuous synthesis of angiogenic growth factors, resulting in rapid neointima formation on the prosthesis after implantation. We expected a similar angiogenic phenomenon to occur if bone marrow was transplanted into ischemic myocardium. Bone marrow was transplanted into ischemic myocardium created in dogs. Marrow cells continued synthesis of angiogenic growth factors, which were effective in protecting the capillary network from ischemia, but not myocytes. Autologous bone marrow was injected intramuscularly into ischemic myocardium created in the left ventricular wall of dogs. Control operations were performed without bone marrow. On days 3 and 7, marrow cells survived, and their adjacent cells and the surrounding extracellular matrix were immunohistochemically bFGF reactive. At 3 weeks, no marrow cells were identified. Myocytes disappeared, but the capillary blood vessel networks remained. With some exceptions, these capillaries did not contain blood cell components. In the controls, scar tissue with a very small number of capillaries was formed. In conclusion, marrow cells survived for a short period of time after transplantation, and continued synthesis of angiogenic growth factors, which were effective in protecting endothelial cells from ischemia, but not myocytes. Therefore, the results also suggest that there are limitations in the treatment of ischemic myocardium using angiogenic growth factors alone.