Design of a composite biomaterial system for tissue engineering applications

Event Abstract Back to Event Molecularly regulated release of growth factors from programmable hydrogels for angiogenesis Yong Wang1*, Nan Zhao1, Xiaolong Zhang1*, Li-Juan Duan2* and Guo-Hua Fong2* 1 Penn State University, Department of Biomedical Engineering, United States 2 UConn Health Center, Center for Vascular Biology, United States Introduction: Vascularization is important to treatment of ischemic diseases and survival of tissue-engineered constructs[1]. Delivery of growth factors is a promising strategy for vascularization. However, polymeric systems for growth factor delivery face several challenges including denaturation of growth factors, difficulty of controlling growth factor release kinetics, and/or lack of macroporous structures for cell growth[2]. The purpose of this work was to demonstrate that oligonucleotide aptamers and macroporous hydrogels could be used to develop a multifunctional biomaterial with great potential of overcoming all of these challenges. Materials and Methods: The multifunctional hydrogel was synthesized in a cylindrical mold with free radical polymerization and gas formation[3]. The key components in the pregel solution were oligonucleotide aptamers and acrylated gelatin that were incorporated into the hydrogel during the polymerization. The incorporation efficiency was determined by protein and oligonucleotide staining assays. The hydrogel was thoroughly washed to remove unreacted monomers and immersed in solution with VEGF and PDGF-BB for growth factor sequestration. The kinetics of growth factor release was examined using ELISA. The bioactivity of released or retained growth factors was measured using the standard HUVEC tube formation assay. To demonstrate directly the ability of this new hydrogel in promoting angiogenesis, the hydrogels with growth factors were subcutaneously implanted into a mouse model. The hydrogels were collected and analyzed after 4-week implantation. Endothelial cell surface biomarker CD31 was stained to examine populated vascular lumens in the hydrogels and tissues adjacent to the hydrogels. Results and Discussion: Macroporous hydrogels were successfully synthesized with free radical polymerization coupled with gas formation. The SEM images show that macroporous structures had an average pore diameter of 50 microns (Figure 1a). Both aptamers and gelatin were efficiently incorporated into the hydrogel network. Importantly, growth factors could be rapidly sequestered into the hydrogel network. When the superporous hydrogels were immersed into release media, aptamers effectively reduced the burst release effect and prolonged the growth factor release with desired kinetics. Moreover, the released and retained growth factors maintained high bioactivity (Figure 1b). For instance, at the end of two weeks, VEGF could maintain ~50% bioactivity that is much higher than the data reported in the literature. The animal studies showed that the multifunctional hydrogel stimulated angiogenesis much more effectively than the control hydrogels without aptamers (Figure 1c). Conclusions: Aptamer-functionalized hydrogels are a promising multifunctional biomaterial for controlled growth factor release. They hold great potential to stimulate vascularization for treatment of ischemic diseases or enhance survival of tissue-engineered constructs. U.S. NSF CAREER program under award number DMR-1332351; the National Heart, Lung, and Blood Institute of the NIH under award number R01HL122311

PURPOSE. Following corneal wounding, early migration of keratocytes into the wound area is of pivotal importance in the healing process, but the nature of this migration is not well understood. The influence of peptide growth factors on the chemotactic and chemokinetic migration of human corneal keratocytes was investigated, using the following growth factors: platelet derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), transforming growth factor-a (TGF-a), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-beta-1 (TGF-ß1). METHODS. The chemotactic stimulation was investigated in the Boyden blind-well chemotaxis chamber, and the chemokinetic effect of the growth factors determined by a modified checkerboard analysis. RESULTS. PDGF-BB, EGF and TGF-ß1 stimulated chemotaxis towards a peak value, with a subsequent decline at higher concentrations. PDGF-BB and EGF peaked at 1 ng/ml with a 2.0 and a 2.5-fold increase respectively in the number of keratocytes migrating, whereas TGF-ß1 reached a maximum response at 0.1 ng/ml, with a 1.7-fold increase. Chemotaxis reached an early plateau and remained constant at concentrations between 1 ng/ml and 100 ng/ml when stimulating with TGF-a (2.7-fold), bFGF (2.0-fold), aFGF (2.7-fold), and IGF-I (4.5-fold). Checkerboard analysis revealed that all growth factors were chemotactic agents for human keratocytes, except bFGF, which principally stimulated chemokinesis. CONCLUSION. These in vitro results demonstrate that PDGF-BB, EGF, TGF-a, aFGF, IGF-I, and TGF-ß1 increase keratocyte chemotaxis, and they may play an important role in the early recruitment of keratocytes to the corneal wound site in vivo.

Nanoparticle-Coated Sutures Providing Sustained Growth Factor Delivery to Improve the Healing Strengths of Injured Tendons

Sustained delivery of growth factors, proteins, drugs and other biologically active molecules is necessary for tissue engineering applications. Electrospun fibers are attractive tissue engineering scaffolds as they partially mimic the topography of the extracellular matrix (ECM). However, they do not provide continuous nourishment to the tissue. In search of a biomimetic scaffold for salivary gland tissue regeneration, we previously developed a blend nanofiber scaffold composed of the protein elastin and the synthetic polymer polylactic-co-glycolic acid (PLGA). The nanofiber scaffold promoted in vivo-like salivary epithelial cell tissue organization and apicobasal polarization. However, in order to enhance the salivary cell proliferation and biomimetic character of the scaffold, sustained growth factor delivery is needed. The composite nanofiber scaffold was optimized to act as a growth factor delivery system using epidermal growth factor (EGF) as a model protein. The nanofiber/EGF hybrid nanofibers were synthesized by double emulsion electrospinning where EGF is emulsified within a water/oil/water (w/o/w) double emulsion system. Successful incorporation of EGF was confirmed using Raman spectroscopy. EGF release profile was characterized using enzyme-linked immunosorbent assay (ELIZA) of the EGF content. Double emulsion electrospinning resulted in slower release of EGF. We demonstrated the potential of the proposed double emulsion electrospun nanofiber scaffold for the delivery of growth factors and/or drugs for tissue engineering and pharmaceutical applications.

The effects of soluble growth factors on regulating the survival and growth of choriocapillaris endothelial (CCE) cells were investigated in vitro. CCE cells were cultured in a serum-free medium in the presence or absence of various soluble growth factors. Cell growth and cell viability were assessed by counting viable cells. Results showed that acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) significantly stimulated CCE growth in a dose-dependent manner. Platelet-derived growth factor BB (PDGF, 0.4–10 ng/ml), insulin-like growth factor (IGF, 0.4–10 ng/ml) or insulin (0.4–10 µg/ml) alone did not affect the growth of CCE cells. In the presence of insulin (10 µg/ml), however, PDGF stimulated CCE growth in a dose-dependent manner. By contrast, transforming growth factor β1 (TGF-β1) induced CCE death in the absence of other growth factors and inhibited the CCE growth induced by aFGF, bFGF or VEGF. When CCE cells had been cultured in media containing aFGF, bFGF, VEGF or the combination of PDGF and insulin for 48 h, withdrawal of aFGF, bFGF or VEGF, but not PDGF, from the media resulted in CCE death. The CCE death induced by either an introduction of TGF-β1 or withdrawal of aFGF, bFGF or VEGF was defined as apoptosis based on morphologic characteristics (condensation and fragmentation of nuclei, shrinkage of cells in size) and DNA fragmentation in multiples of approximately 180 base pairs. Phorbol 12-myristate 13-acetate (PMA, 2 or 5 nM) protected CCE cells against apoptosis induced by the introduction of TGF-β1 and withdrawal of aFGF, bFGF or VEGF, while H7 (50 µM), but not HA1004 (50 µM), abrogated the protective effect of PMA on CCE apoptosis. However, cycloheximide (0.1 µM), a protein synthesis inhibitor, was only effective to protect CCE cells from apoptosis induced by aFGF, bFGF or VEGF withdrawal, but not that induced by the introduction of TGF-β1. Moreover, cycloheximide and a low concentration of PMA (2 nM) showed an additive effect on protecting CCE cells from apoptosis as the result of a growth factor withdrawal. These data may suggest that some growth factors, such as aFGF, bFGF, VEGF and TGF-β1, play a more critical role than the others, such as PDGF and IGF, in the regulation of CCE growth and survival. CCE apoptosis as a result of withdrawal of a survival factor or of receiving a death factor such as TGF-β1 may involve different mechanisms, which merits further study.

The rationale for using topical platelet gel therapy is to provide the healing tissues with concentrated platelet-derived factors. Several systems are available to prepare platelet-rich plasma (PRP) and from these, the platelet gel. These systems produce two- to six-fold platelet and growth factor-enriched concentrations. The bioavailability of growth factors in tissue healing depends on the amount of growth factors stored in platelets but a portion of these is lost during platelet manipulation. Very few data have been reported on the kinetics of growth factor release from PRP-gels. The aim of this study is to assess the growth factor recovery and its bioavailability to tissues in four different PRP and PRP-gel preparation techniques. Three commercially available devices (Fibrinet, RegenPRP-Kit, Plateltex) and one manual procedure (home made, HM) were evaluated with reference to resulting platelet concentration, growth factor content and the kinetics of growth factor release from gel. Platelet concentration increased from 1.65- to 4.4-fold in comparison with whole blood initially used. The final platelet concentration (x 10(3)/microl) was: Fibrinet 1358 +/- 419, Regen 430 +/- 109, HM 1196 +/- 188, and Plateltex 1160 +/- 164. A high variation (5- to 27-fold) was found in growth factor concentration in relation to the method used and also a high variation in the kinetics of growth factor release from gels. Similar methods for platelet gel preparation revealed different performances concerning growth factor recovery and the kinetics of its release from the gel. It is unclear whether these noticeable differences are important for clinical management.

Vasculogenesis 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.

In vitro comparative evaluation of recombinant growth factors for tissue engineering of bladder in patients with neurogenic bladder

Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair

Emerging growth factor delivery hydrogel system for wound healing

BackgroundClinical application of platelet-rich-plasma (PRP) has been accelerated to investigate early recovery from various musculoskeletal conditions. It involves the promotion of tissue damage repair through the action of multiple growth factors at physiological concentrations. The composition of PRP differs based on many factors, which may include age and gender. Therefore, we analyzed correlations between age, gender, and platelet counts in PRP with growth factors in Japanese subjects.MethodPeripheral blood was drawn from 39 healthy volunteers between 20 and 49 years of age (age, mean ± standard deviation = 33 ± 8.7 years; gender ratio, male:female = 19:20; BMI, mean ± standard deviation = 22 ± 4.0) and prepared through centrifugation (volume, 6 mL per sample). After being activated with CaCl2, the supernatant was stored. The mean platelet count in PRP was 41.4 ± 12.2 × 104/μL. PRP concentration rate (i.e., PRP/peripheral platelet counts) was 1.8 ± 0.4 times. Growth factor levels (platelet-derived growth factor-BB, transforming growth factor-β1, vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factor-1, and hepatocyte growth factor) were measured using enzyme-linked immunosorbent assay (ELISA), and correlations with age, gender, and PRP platelet counts were statistically analyzed by calculating Spearman’s rank correlation coefficients (r).ResultsAge was negatively correlated with platelet-derived growth factor-BB and insulin-like growth factor-1 (r = − 0.32, − 0.39), and gender had no influence on growth factors. Platelet counts in PRP positively correlated with platelet-derived growth factor-BB, transforming growth factor-β1, epidermal growth factor, and hepatocyte growth factor (r = 0.39, 0.75, 0.71, and 0.48, respectively).ConclusionsThis clinical study shows a significant variation of PRP among individual patients and that this variation is influenced by the age and the platelet counts of the subjects. Our data demonstrate that patient characteristics account for the differences in PRP physiological activity.

Gene therapy is a promising strategy to deliver growth factors of interest locally in a sustained fashion and has the potential to overcome barriers to using recombinant protein therapy such as sustainability and cost. Recent studies demonstrate the safety and efficacy of non-viral delivery of plasmid DNA (pDNA) encoding a single growth factor to enhance bone healing. This pilot study is aimed at testing a non-viral gene delivery system that can deliver two different plasmids encoding two different growth factors. Polyethylenimine (PEI), a cationic polymer, was utilized as a gene delivery vector and collagen scaffold was used as a carrier to deliver the PEI-pDNA complexes encoding platelet derived growth factor B (PDGF-B) and/or vascular endothelial growth factor (VEGF). Calvarial defects in rats were implanted with scaffolds containing PEI-pPDGF-B complexes, PEI-pVEGF complexes or containing both PEIpPDGF- B and PEI-pVEGF complexes in a 1:1 ratio of plasmids. The results indicated that bone regeneration as measured using micro-CT and histological assessments was inferior in groups treated with PEI-(pPDGF-B + pVEGF) complexes, compared to defects treated with PEI-pPDGF-B complexes. This pilot study that explores the feasibility and efficacy of combinatorial non-viral gene delivery system for bone regeneration appears to provide a rationale for investigation of sequential delivery of growth factors at specific time points during the healing phases and this will be explored further in future studies.

Biomaterials based growth factor delivery for brain regeneration after injury

Background The role of growth factors in prosthesis loosening is unclear. We evaluated the levels of plate-let-derived growth factor BB (PDGF-BB), transforming growth factors β1 (TGF-β1) and β2 (TGF-β2), both before and after activation, in patients with aseptic loosening of their hip prosthesis.Patients and methods 26 patients with loosened hip implants were compared with 21 patients who had stable hip prostheses, and 28 patients undergoing primary hip replacement. The plasma levels of the growth factors were analyzed by enzyme immunoassay. TGF-β1 and TGF-β2 were determined both before and after activation.Results Patients with aseptic loosening had significantly lower PDGF-BB levels than patients undergoing primary hip replacement, and significantly lower TGF-β2 levels than patients with a stable implant. Patients with stable prostheses had significantly higher TGF-β1 and TGF-β2 levels than patients undergoing primary hip replacement.Interpretation It is possible that the prosthetic implant itself causes a local increase in PDGF-BB, TGF-β1 and TGF-β2, released by osteoblasts and other cells in the microenvironment. The plasma PDGF-BB measured does not correspond to local release, which is probably due to local consumption or degradation. The consumption of PDGF-BB is low in stable implants, and TGF-β1 and TGF-β2 levels increase during bone formation. In loosening, PDGF-BB consumption is higher and causes a significant reduction in plasma levels as compared to presurgery. The formation of poor-quality bone may be related to the scarce increase in TGF-β1 and TGF-β2. In conclusion, compared with patients with a stable implant, a reduction in bone-forming growth factors appears to occur in individuals with aseptic loosening.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.