Delivery Of Multiple Growth Factors Research Articles

How Does Temporal and Sequential Delivery of Multiple Growth Factors Affect Vascularization Inside 3D Hydrogels?

AbstractVasculogenesis and angiogenesis are leveraged by orchestrated secretion of several growth factors. Mimicking this process in vitro can maximize vascularization inside 3D cell cultures. While the role of individual growth factors, such as vascular endothelial growth factor, Ephrin‐B2, angiopoietins, and platelet‐derived growth factor‐BB (PDGF‐BB) is well studied, the temporal influence of growth factor secretion, and the effect of their concentrations and combinations on in vitro vascularization inside hydrogels, have not been systematically investigated. Here, combinations of angiopoietin‐1, angiopoietin‐2, and PDGF‐BB improve vascularization of a human umbilical vein endothelial cells‐fibroblast coculture inside polyethylene glycol‐based hydrogels the most, while the optimal concentrations and time points of growth factor addition are determined. Moreover, fibroblasts, pericytes, and mesenchymal stem cells (MSCs) are compared as supporting cells, of which MSCs best promote vascularization in coculture. Additionally, the resulting blood vessels align with magnetically oriented rod‐shaped microgels when cultured inside the Anisogel. To mimic fibrosis, transforming growth factor‐beta is added, resulting in significantly smaller vessels and more collagen secretion. This in vitro study reveals that a cascade of growth factors can improve vascular formation in 3D hydrogels, which is important to create viable tissue‐engineered constructs for therapies and in vitro healthy and diseased tissue models.

Enzyme-Mediated Nerve Growth Factor Release from Nanofibers Using Gelatin Microspheres.

Spinal cord injury is a complex environment, with many conflicting growth factors present at different times throughout the injury timeline. Delivery of multiple growth factors has received mixed results, highlighting a need to consider the timing of delivery for possibly antagonistic growth factors. Cell-mediated degradation of delivery vehicles for delayed release of growth factors offers an attractive way to exploit the highly active immune response in the spinal cord injury environment. In this study, growth factor-loaded gelatin microspheres (GMS) combined with methacrylated hyaluronic acid (MeHA) were electrospun to create GMS fibers (GMSF) for delayed release of growth factors (GFs). GMS were successfully combined with MeHA while electrospinning, with an average fiber diameter of 365 ± 10 nm and 44% ± 8% fiber alignment. GMSF with nerve growth factor (NGF) was tested on dissociated chick dorsal root ganglia cells. We further tested the effect of M1 macrophage-conditioned media (M1CM) to simulate macrophage invasion after spinal cord injury for cell-mediated degradation. We hypothesized that neurons grown on GMSF with loaded NGF would exhibit longer neurites in M1CM, showing a release of functional NGF, as compared with controls. GMSF in M1CM was significantly different from MeHA in serum-free media (SFM) and M0-conditioned media (M0CM), as well as GMSF in M0CM (p < 0.05). Moreover, GMSF + NGF in all media conditions were significantly different from MeHA in SFM and M0CM (p < 0.05). The goal of this study was to develop a biomaterial system where drug delivery is triggered by immune response, allowing for more control and longer exposure to encapsulated drugs. The spinal cord injury microenvironment is known to have a robust immune response, making this immune-medicated drug release system particularly significant for directed repair.

A safety evaluation of allogeneic freeze-dried platelet-rich plasma or conditioned serum compared to autologous frozen products equivalents in equine healthy joints

BackgroundHemoderivatives such as autologous conditioned serum (ACS) and platelet-rich plasma (PRP) have been used as potential disease-modifying therapies in musculoskeletal disorders such as osteoarthritis (OA). These therapies are based on the delivery of multiple growth factors and anti-inflammatory cytokines that are known to participate in inflammatory processes. The variability of cytokine content due to the autologous nature of the product, the non-availability for immediate use and need for storage at low temperatures are limitations for its use in the field. An allogeneic freeze-dried conditioned serum (CS) and PRP would provide field clinicians with a more practical approach to use such products in daily practice. Based on in vitro preliminary data, this experimental study aimed to test the in vivo safety of allogeneic freeze-dried CS and PRP in healthy joints, using the horse as a model.ResultsEight horses were randomly assigned and treated with PRP or CS. Horses had three joints injected with ALLO-FD PRP or CS, and three contralateral joints injected with the AUTO version of the same product, by a blinded clinician. Horses were evaluated clinically, and had synovial fluid collected at different time points and evaluated for cell content, PGE2 and protein. Both CS and PRP products triggered a self-limiting and mild inflammatory response in equine healthy joints. This was indicated by the transient increase in nucleated cell count, PGE2 and total protein in synovial fluid. This mild inflammatory response did not result in significant lameness and was not different among the groups.ConclusionsThe allogeneic freeze-dried PRP and CS showed to be overall safe and not dissimilar compared to their autologous frozen version in equine healthy joints. Further studies are necessary to evaluate the modulatory effects of these therapies in a clinical setting.

Control Delivery of Multiple Growth Factors to Actively Steer Differentiation and Extracellular Matrix Protein Production.

In tissue engineering, biomaterials have been used to steer the host response. This determines the outcome of tissue regeneration, which is modulated by multiple growth factors (GFs). Hence, a sustainable delivery system for GFs is necessary to control tissue regeneration actively. A delivery technique of single and multiple GF combinations, using a layer-by-layer (LBL) procedure to improve tissue remodeling, is developed. TGF-β1, PDGF-ββ, and IGF-1 are incorporated on tailor-made polymeric rods, which could be used as a tool for potential tissue engineering applications, such as templates to induce the formation of in situ tissue engineered blood vessels (TEBVs). Cell response is analyzed in vitro using rat and human dermal fibroblasts for cellular proliferation, fibroblast differentiation, and extracellular matrix (ECM) protein synthesis. Results revealed a higher loading efficiency and control release of GFs incorporated on chloroform and oxygen plasma-activated (COX) rods. Single PDGF-ββ and IGF-1 release, and dual release with TGF-β1 from COX rods, showed higher cell proliferation when compared to COX rods alone. A substantial increase in α-smooth muscle actin (α-SMA) is also observed in GF releasing COX rods, with TGF-β1 COX rods providing the most pronounced differentiation. A significant increase in collagen and elastin synthesis is observed on all GF releasing COX rods compared to control, with COX rods releasing TGF-β1 and IGF-1 providing the highest secretion. TGF-β1 and IGF-1 releasing COX rods induced higher Glycosaminoglycan (GAG)/DNA amounts than the other GF releasing COX rods. As PDGF-ββ and TGF-β1/PDGF-ββ COX rods displayed the highest fibroblast attachment, these rods provided the highest total collagen and elastin production. The attractive results from efficiently incorporating single and multiple GFs on COX rods and their sustainable release to steer cellular behavior suggest a promising route to enrich the formation of in situ engineered tissues.

In vitro characterisation of 3D printed platelet lysate-based bioink for potential application in skin tissue engineering

Wounds impact millions of patients every year and represent a serious cause of morbidity and mortality worldwide, yet current treatment outcomes are far from ideal. Therapies based on delivery of multiple growth factors offer a promising approach for optimal wound management; however, their high production cost, low stability, and lack of effective delivery system limits their application in the clinic. Platelet lysate is a suitable, abundant and cost-effective source of growth factors that play an important role in the healing cascade. The aim of this current work is to develop an extrusion-based bioink consisting of platelet lysate (PL) and gelatin methacryloyl (GelMA) (PLGMA) for the fabrication of a multifunctional 3D printed dermal equivalent. This bioink meets the essential requirements of printability in terms of rheological properties and shape fidelity. Moreover, its mechanical properties can be readily tuned to achieve stiffness that is equivalent to native skin tissue. Biologically relevant factors were successfully released in a sustainable manner for up to two weeks of study. The bioavailability of those factors was demonstrated by high cell viability, good cell attachment and improved proliferation of printed dermal fibroblasts. Furthermore, growth factors upregulated ECM synthesis and deposition by dermal fibroblasts after two weeks of culture.

Thermosensitive heparin-poloxamer hydrogel encapsulated bFGF and NGF to treat spinal cord injury.

The application of growth factors (GFs) for treating chronic spinal cord injury (SCI) has been shown to promote axonal regeneration and functional recovery. However, direct administration of GFs is limited by their rapid degradation and dilution at the injured sites. Moreover, SCI recovery is a multifactorial process that requires multiple GFs to participate in tissue regeneration. Based on these facts, controlled delivery of multiple growth factors (GFs) to lesion areas is becoming an attractive strategy for repairing SCI. Presently, we developed a GFs‐based delivery system (called GFs‐HP) that consisted of basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and heparin‐poloxamer (HP) hydrogel through self‐assembly mode. This GFs‐HP was a kind of thermosensitive hydrogel that was suitable for orthotopic administration in vivo. Meanwhile, a 3D porous structure of this hydrogel is commonly used to load large amounts of GFs. After single injection of GFs‐HP into the lesioned spinal cord, the sustained release of NGF and bFGF from HP could significantly improve neuronal survival, axon regeneration, reactive astrogliosis suppression and locomotor recovery, when compared with the treatment of free GFs or HP. Moreover, we also revealed that these neuroprotective and neuroregenerative effects of GFs‐HP were likely through activating the phosphatidylinositol 3 kinase and protein kinase B (PI3K/Akt) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signalling pathways. Overall, our work will provide an effective therapeutic strategy for SCI repair.

Degradable Poly(Methyl Methacrylate)-co-Methacrylic Acid Nanoparticles for Controlled Delivery of Growth Factors for Bone Regeneration.

Bone tissue engineering strategies have been developed to address the limitations of the current gold standard treatment options for bone-related disorders. These systems consist of an engineered scaffold that mimics the extracellular matrix and provides an architecture to guide the natural bone regeneration process, and incorporated growth factors that enhance cell recruitment and ingress into the scaffold and promote the osteogenic differentiation of stem cells and angiogenesis. In particular, the osteogenic growth factor bone morphogenetic protein 2 (BMP-2) has been widely studied as a potent agent to improve bone regeneration. A key challenge in growth factor delivery is that the growth factors must reach their target sites without losing bioactivity and remain in the location for an extended period to effectively aid in the formation of new bone. Protein incorporation into nanoparticles can both protect protein bioactivity and enable its sustained release. In this study, a poly(methyl methacrylate-co-methacrylic acid) nanoparticle-based system was synthesized incorporating a custom poly(ethylene glycol) dimethacrylate crosslinker. It was demonstrated that the nanoparticle degradation rate can be controlled by tuning the number of hydrolytically degradable ester units along the crosslinker. We also showed that the nanoparticles had high affinity for a model protein for BMP-2, and optimal conditions for maximum protein loading efficiency were elucidated. Ultimately, the proposed system and its high degree of tunability can be applied to a wide range of growth factors and tissue engineering applications. Impact Statement In this study, we developed a novel method of synthesizing nanoparticles with tunable degradation rates through the incorporation of a custom synthesized, hydrolytically degradable crosslinker. In addition, we demonstrated the affinity of the synthesized nanoparticles for a model protein for bone morphogenetic protein 2 (BMP-2). The tunability of these nanoparticles can be used to develop complex tissue engineering systems, for example, for the delivery of multiple growth factors involved at different stages of the bone regeneration process.

Surface functionalization of dual growth factor on hydroxyapatite-coated nanofibers for bone tissue engineering

To improve bone growth and to imitate the function of natural extracellular matrix for sustained release of multiple growth factors, a scaffold of porous gelatin nanofibers were electrospun, coated with hydroxyapatite using a simulated body fluid solution, and surface-functionalized with avidin to facilitate binding with biotinylated growth factors, namely bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2) at different ratios. Scanning electron microscopy was used for structure characterization and Fourier-transform infrared spectroscopy and calcium assay were performed to evaluate the calcium composition. Immunostaining techniques confirmed the conjugation of multiple growth factors. The effects of the two growth factors and hydroxyapatite on osteogenic cell differentiation were studied using quantitative polymerase chain reaction analysis. Multiple growth factors were successfully conjugated onto the functionalized surface by controlling the FGF-2/BMP-2 ratio. Comparisons of the factor release profiles with those of the physical adsorption showed that avidin-biotin conjugation was more effective for sustained release. Bone regeneration was enhanced via synergism between multiple growth factor delivery and the hydroxyapatite nanofiber coating, as confirmed by the increased expression of osteogenic gene markers. The nanofibers thus provide a promising osteoconductive scaffold with controlled multiple growth factor delivery for bone tissue engineering.

A Comprehensive Review of Concentrated Growth Factors and Their Novel Applications in Facial Reconstructive and Regenerative Medicine.

Concentrated growth factors (CGFs) are the latest generation of platelet concentrates. The objective of developing CGF is to increase therapeutic efficacy. However, few studies have supported the superiority of CGF in composition and efficacy. The reconstruction and regeneration process is complicated and long term, whereas bioactivity of CGF is not durable. The purpose of this review is threefold. The first is to recommend more comparative studies between CGF and other platelet concentrates. The second is to constitute a continuous drug delivery system by combining CGF with other biomaterials. Finally, the novel use of CGF in facial regenerative and reconstructive medicine will be highlighted. A comprehensive review of literature regarding the use of CGF in facial regenerative and reconstructive medicine was performed. Based on the inclusion and exclusion criteria, a total of 135 articles were included. The use of CGF involving facial rejuvenation, cartilage grafting, facial bone defects, facial peripheral nerve injury and wounding is reviewed. The reconstructive and regenerative principles lie in firm fibrin scaffolds and continuous in situ delivery of multiple growth factors. CGF represents an advance in personalized medicine concept. However, the current scientific evidences about the use of CGF are limited. More basic and clinical studies should be conducted to understand the characteristics and clinical application of CGF. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Dual growth factor delivery using PLGA nanoparticles in silk fibroin/PEGDMA hydrogels for articular cartilage tissue engineering.

Degeneration of articular cartilage due to damages, diseases, or age-related factors can significantly decrease the mobility of the patients. Various tissue engineering approaches which take advantage of stem cells and growth factors in a three-dimensional constructs have been used for reconstructing articular tissue. Proliferative impact of basic fibroblast growth factor (bFGF) and chondrogenic differentiation effect of transforming growth factor-beta 1 (TGF-β1) over mesenchymal stem cells have previously been verified. In this study, silk fibroin (SF) and of poly(ethylene glycol) dimethacrylate (PEGDMA) were used to provide a versatile platform for preparing hydrogels with tunable mechanical, swelling and degradation properties through physical and chemical crosslinking as a microenvironment for chondrogenic differentiation in the presence of bFGF and TGF-β1 releasing nanoparticles (NPs) for the first time. Scaffolds with compressive moduli ranging from 95.70 ± 17.82 to 338.05 ± 38.24 kPa were obtained by changing both concentration PEGDMA and volume ratio of PEGDMA with 8% SF. Highest cell viability was observed in PEGDMA 10%-SF 8% (1:1) [PEG10-SF8(1:1)] hydrogel group. Release of bFGF and TGF-β1 within PEG10-SF8(1:1) hydrogels resulted in higher DNA and glycosaminoglycans amounts indicating synergistic effect of dual release over proliferation and chondrogenic differentiation of dental pulp stem cells in hydrogels, respectively. Our results suggested that simultaneous delivery of bFGF and TGF-β1 through utilization of PLGA NPs within PEG10-SF8(1:1) hydrogel provided a novel and versatile means for articular cartilage regeneration as they allow for dosage- and site-specific multiple growth factor delivery.

Use of Nanoparticles in Tissue Engineering and Regenerative Medicine.

Advances in nanoparticle (NP) production and demand for control over nanoscale systems have had significant impact on tissue engineering and regenerative medicine (TERM). NPs with low toxicity, contrasting agent properties, tailorable characteristics, targeted/stimuli-response delivery potential, and precise control over behavior (via external stimuli such as magnetic fields) have made it possible their use for improving engineered tissues and overcoming obstacles in TERM. Functional tissue and organ replacements require a high degree of spatial and temporal control over the biological events and also their real-time monitoring. Presentation and local delivery of bioactive (growth factors, chemokines, inhibitors, cytokines, genes etc.) and contrast agents in a controlled manner are important implements to exert control over and monitor the engineered tissues. This need resulted in utilization of NP based systems in tissue engineering scaffolds for delivery of multiple growth factors, for providing contrast for imaging and also for controlling properties of the scaffolds. Depending on the application, materials, as polymers, metals, ceramics and their different composites can be utilized for production of NPs. In this review, we will cover the use of NP systems in TERM and also provide an outlook for future potential use of such systems.

Winner of the society for biomaterials student award in the Ph.D. category for the annual meeting of the society for biomaterials, april 11-14, 2018, Atlanta, GA: Development of a bimodal, in situ crosslinking method to achieve multifactor release from electrospun gelatin.

To better mimic native tissue microenvironments, current efforts have moved beyond single growth factor delivery to more complex multiple growth factor delivery with distinct release profiles. Electrospun gelatin, a widely investigated drug delivery vehicle, requires postprocessing crosslinking techniques that generate a mesh with uniform crosslinking density, limiting the ability to deliver multiple factors at different rates. Herein, we describe a method to independently control release of multiple factors from a single electrospun gelatin mesh. Two in situ crosslinking modalities, photocrosslinking of methacyrlated gelatin and reactive crosslinking of gelatin with a diisocyanate, are coelectrospun to generate distinct fiber populations with different crosslinking chemistry and density in a single mesh. The photocrosslinked gelatin-methacrylate resulted in a relatively rapid release of a model protein (48 ± 12% at day 1, 96 ± 3% at day 10) due to diffusion of embedded protein from the crosslinked fibers. The reactive crosslinking system displayed a more sustained release (7 ± 5% at day 1, 33 ± 2% at day 10) that was attributed to the conjugation of protein to gelatin with the diisocyanate, requiring degradation of gelatin prior to diffusion out of the fibers. Both modalities displayed tunable release profiles. Subsequent release studies of a cospun mesh with two different crosslinked fiber populations confirmed that the cospun mesh displayed multifactor release with independent release profiles. Overall, this bimodal, in situ crosslinking approach enables the delivery of multiple factors with distinct release kinetics from a single mesh and is expected to have broad utility in tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1155-1164, 2018.

Bone morphogenetic protein-2 associated multiple growth factor delivery for bone tissue regeneration

Administration of growth factors (GFs) and cytokines to damaged bone tissue for the purpose of bone tissue regeneration has been investigated. Among GFs and cytokines, bone morphogenetic protein-2 (BMP-2) is one of the most important stimulatory factors for enhancing the osteoblastic differentiation of progenitor cells and promoting bone formation. Co-delivery of other GFs, including vascular endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and stromal cell-derived factor, with BMP-2 is known to facilitate repair and remodeling of damaged bone tissue. These GFs stimulate various cellular activities, including angiogenesis, migration, proliferation, regulation of proteoglycan level, and stem cell recruitment. Although bone repair and regeneration are dynamic and complex physiological processes involving multiple cell types and cytokines in timed healing cascade stages, BMP-2-associated delivery of dual and/or multiple GFs could promote bone tissue regeneration based on the synergistic effects of GFs compared with single GF treatment. Current engineering approaches emphasize the importance of design parameters for GF delivery systems, such as optimization of the release profiles, stabilities, and bioactivities of cargo GFs, and the physico-chemical properties of scaffold/hydrogel materials. Therefore, this review summarizes BMP-2-associated GF combinations, biological functions, and current delivery strategies utilized in bone tissue engineering for the development of feasible clinical applications.

Multiple growth factor delivery for skin tissue engineering applications

Administration of exogenous growth factors (GFs) to a damaged site has been investigated for skin tissue regeneration. Among the many types of GFs and cytokines, epidermal growth factor, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, and hepatocyte growth factor could be specifically used for stimulating molecules in wound healing as well as for recovery of damaged skin tissues. It is speculated that delivered GFs could stimulate various cellular functions, including proliferation, migration, deposition of extracellular matrix molecules, and remodeling of collagen synthesis. Although the physiological wound healing process is complex, engineering strategies for proper delivery of multiple therapeutic GFs could enhance the quality and quantity of regenerated skin tissues. As compared to single delivery of a GF, recent studies have proven that any combination of multiple GFs and/or therapeutic chemical factors synergistically facilitates the regeneration of damaged skin tissues. In order to maximize the stability, bioactivity, intrinsic therapeutic functionality, and efficiency of internal delivery of cargo GFs, it is essential to utilize tissueengineered biomaterials and related composites as implantable platforms. Successful fabrication and development of skin tissue engineering applications as well as subsequent surgical implantation of these platforms might provide clinical treatment for superior skin regeneration. Therefore, the present review summarizes the biological functions, related signaling mechanisms, and recent developments of tissue engineering applications for multiple GF delivery.

Sequential delivery of VEGF, FGF-2 and PDGF from the polymeric system enhance HUVECs angiogenesis in vitro and CAM angiogenesis

Angiogenesis is an organized series of events, beginning with vessel destabilization, followed by endothelial cell re-organization, and ending with vessel maturation. The formation of a mature vascular network requires precise spatial and temporal regulation of a large number of angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor (PDGF). VEGF aids in vascular permeability and endothelial cell recruitment, FGF-2 activates endothelial cell proliferation and migration while PDGF stimulates vascular stability. Accordingly, VEGF may inhibit vessel stabilization while PDGF may inhibit endothelial cell recruitment. Therefore, a new polymeric system was prepared by the supercritical carbon dioxide foaming technology, which realized sequential delivery of two or more growth factors with the controlled dose and rate. Increased release of VEGF (71.10%) and FGF-2 (69.76%) compared to PDGF (43.17%) was observed for the first 7 days. Thereafter, up till 21 days, an increased rate of release of BMP-2 compared to VEGF 165 was observed. The effects of PDGF-PLAms/VEGF-FGF-2-PLGA scaffolds on angiogenesis were investigated by human umbilical vein endothelial cells (HUVECs) angiogenic differentiation in vitro and chorioallantoic membrane (CAM) angiogenesis in vivo. Sequential delivery of VEGF, FGF-2 and PDGF from structural polymer scaffolds with distinct kinetics resulted in significant angiogenic differentiation of HUVECs and rapid formation of mature vascular networks in chorioallantoic membrane. This study reported a composite scaffold with distinct release kinetics, and these results clearly indicated the importance of sequential delivery of multiple growth factors in tissue regeneration and engineering.

Development of multifunctional films for peripheral nerve regeneration.

This manuscript focuses on the development of multifunctional degradable polymer films that provide topographic cues for guided growth, surface gradients of growth factors as well as nanoparticles in the films for tunable release of growth factors to enable peripheral nerve regeneration. The combination of cues was designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. These multifunctional films successfully provided high protein loading capacities while persevering activity, protein gradients on the surface, and tunable release of bioactive nerve growth factor that promoted directional and guided neurite extension of PC12 cells of up to 10μm in 2days. These multifunctional films can be made into conduits for peripheral nerve regeneration.

Muscle Tissue Engineering Using Gingival Mesenchymal Stem Cells Encapsulated in Alginate Hydrogels Containing Multiple Growth Factors.

Repair and regeneration of muscle tissue following traumatic injuries or muscle diseases often presents a challenging clinical situation. If a significant amount of tissue is lost the native regenerative potential of skeletal muscle will not be able to grow to fill the defect site completely. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material, present an advantageous alternative therapeutic option for muscle tissue engineering in comparison to current treatment modalities available. To date, there has been no report on application of gingival mesenchymal stem cells (GMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of the current study were to develop an injectable 3D RGD-coupled alginate scaffold with multiple growth factor delivery capacity for encapsulating GMSCs, and to evaluate the capacity of encapsulated GMSCs to differentiate into myogenic tissue in vitro and in vivo where encapsulated GMSCs were transplanted subcutaneously into immunocompromised mice. The results demonstrate that after 4weeks of differentiation in vitro, GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited muscle cell-like morphology with high levels of mRNA expression for gene markers related to muscle regeneration (MyoD, Myf5, and MyoG) via qPCR measurement. Our quantitative PCR analyzes revealed that the stiffness of the RGD-coupled alginate regulates the myogenic differentiation of encapsulated GMSCs. Histological and immunohistochemical/fluorescence staining for protein markers specific for myogenic tissue confirmed muscle regeneration in subcutaneous transplantation in our in vivo animal model. GMSCs showed significantly greater capacity for myogenic regeneration in comparison to hBMMSCs (p<0.05). Altogether, our findings confirmed that GMSCs encapsulated in RGD-modified alginate hydrogel with multiple growth factor delivery capacity is a promising candidate for muscle tissue engineering.

Calcium phosphate/polyelectrolyte multilayer coatings for sequential delivery of multiple growth factors

Event Abstract Back to Event Calcium phosphate/polyelectrolyte multilayer coatings for sequential delivery of multiple growth factors Emily Jacobs1, 2, Marja Hurley3, Gloria Gronowicz3 and Liisa T. Kuhn1, 2 1 UConn, Biomedical Engineering, United States 2 UConn Health, Reconstructive Sciences, United States 3 UConn Health, School of Medicine, United States Introduction: Many of the most promising strategies for regenerative tissue-engineering aim to replicate natural cellular microenvironments using biomimetic, resorbable materials capable of growth factor delivery. Combinations of growth factors synergistically enhance tissue regeneration[1], but require sequential, rather than co-delivery delivery for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings retain growth factor activity, however, staggered, sequential delivery from these coatings requires barrier layers to prevent interlayer diffusion of multiple factors that results in co-delivery. Here we demonstrate that the incorporation of a biomimetic calcium phosphate (bCaP) layer into PEM can prevent interlayer diffusion of biomolecules and enable staggered delivery. This modified PEM system is one of few aqueous, low temperature systems available for sequential, multifactor delivery and has potential to significantly impact the regenerative tissue-engineering field. Methods: bCaP coatings were prepared on sandblasted, 22 mm, treated tissue culture plastic disks, (NUNC, Rochester, NY) using a simulated body fluid method[2]. The PEM coatings (8-30 bilayers) were applied on top of the bCaP layer by automated alternate 10 min dipping into 1 mg/ml poly L glutamic acid (-) or poly-L lysine (+) solutions (Sigma, St. Louis, MO) with saline rinses between. Coatings were characterized with x-ray diffraction and scanning electron microscopy. To demonstrate sequential delivery a combination of a proliferative factor, recombinant human fibroblast growth factor-2 (FGF-2) (150 ng/disk) (R & D Systems, Minneapolis, MN), and a cytotoxic factor antimycin A (AntiA) (213 μg/disk)(Sigma, St. Louis, MO) was used. In vitro cellular activity on the prepared coatings was assessed with MC3T3-E1 mouse calvarial osteoprogenitor cells (ATCC, Manassas, VA) seeded at 10 or 40 x 103 cells/cm2. Proliferative and cytotoxic effects were quantified using the LIVE/DEAD® assay (Invitrogen Life Technologies, Grand Island, NY). Statistical significances were determined by unpaired t-tests and one-way ANOVA with Tukey post-tests. Results: Uniform nanocrystalline coatings of bCaP (Fig. 1A) and bCaP plus 30 bilayers of PEM (CaP-PEM30) (Fig. 1B) on the disks were observed via SEM. MC3T3-E1s cultured directly on adsorbed AntiA with no bCaP or PEM coating (AntiA) resulted in 90% cell death on day 1 (Fig. 2A). MC3T3-E1s cultured on AntiA embedded under 8 bilayers of PEM (AntiA-PEM8) resulted in 47% cell death on day 1 due to diffusion of AntiA through PEM8. MC3T3-E1s cultured on AntiA embedded under bCaP and 8 bilayers of PEM (AntiA-CaP-PEM8) resulted in 15% cell death, significantly better than PEM only (p<0.001). These data demonstrate that the addition of bCaP to PEM coatings inhibits interlayer diffusion. Active FGF-2 was delivered from 30 bilayers of PEM above CaP (CaP-PEM30-FGF2) as demonstrated by a significant increase in MC3T3-E1 proliferation on day 1 as compared to cells cultured on the FGF-2-free control, (Fig. 2B). No growth factor release was detected by ELISA without cells present. Sequential access of cells to FGF-2 followed by AntiA was also demonstrated (Fig. 3A). On day 1, no differences were observed between CaP-PEM30-FGF2 and AntiA-CaP-PEM30-FGF2 indicating access to AntiA was successfully blocked by bCaP. On day 5 however, cell-mediated degradation of PEM30 and bCaP resulted in a significant decrease in LIVE staining between groups (Fig. 3A) indicating AntiA delivery. Conclusions: Addition of a bCaP barrier layer to a PEM delivery system prevents unwanted interlayer diffusion of embedded factors and enables cell-mediated sequential delivery of multiple factors. This technology can be utilized in multiple research applications where a sequential delivery profile activated by cell degradation of matrix is desired. Funding provided from NIH NIDCR R01DE021103

The scope and sequence of growth factor delivery for vascularized bone tissue regeneration

Bone regeneration is a complex process, that in vivo, requires the highly coordinated presentation of biochemical cues to promote the various stages of angiogenesis and osteogenesis. Taking inspiration from the natural healing process, a wide variety of growth factors are currently being released within next generation tissue engineered scaffolds (in a variety of ways) in order to heal non-union fractures and bone defects. This review will focus on the delivery of multiple growth factors to the bone regeneration niche, specifically 1) dual growth factor delivery signaling and crosstalk, 2) the importance of growth factor timing and temporal separation, and 3) the engineering of delivery systems that allow for temporal control over presentation of soluble growth factors. Alternative methods for growth factor presentation, including the use of gene therapy and platelet-rich plasma scaffolds, are also discussed.

Controlled release of rhEGF and rhbFGF from electrospun scaffolds for skin regeneration.

Controlled delivery of multiple therapeutic agents can be considered as an effective approach in skin tissue engineering. In this study, recombinant human epidermal growth factor (rhEGF) and recombinant human basic fibroblast growth factor (rhbFGF) encapsulated in PLGA microspheres were loaded in hybrid scaffolds of PLGA and PEO. The scaffolds with various formulations were fabricated through electrospinning in order to maintain dual, individual or different release rate of rhEGF and rhbFGF. Morphological, physical and mechanical properties of the scaffold were investigated. The scaffold possessed uniform morphology with an average diameter of 280 nm for PLGA and 760 nm for PEO nanofibers. Furthermore, the mechanical properties of the scaffolds were shown to be akin to those of human skin. Bioactivity of the scaffolds for human skin fibroblasts was evaluated. The HSF acquired significant proliferation and well-spread morphology on the scaffolds particularly in the case of different release rate of rhEGF and rhbFGF which implies the synergistic effect of the growth factors. Additionally, collagen and elastin gene expression was significantly up-regulated in the HSF seeded on the scaffolds in the case of individual delivery of rhEGF and dual delivery of rhEGF and rhbFGF. In conclusion, the prepared scaffolds as a suitable supportive substrate and multiple growth factor delivery system can find extensive utilization in skin tissue engineering.