RGD Hydrogels Research Articles

Engineering a 3D Biomimetic Peptides Functionalized-Polyethylene Glycol Hydrogel Model Cocultured with Endothelial Cells and Astrocytes: Enhancing In Vitro Blood-Brain Barrier Biomimicry.

The blood-brain barrier (BBB) poses a significant challenge for drug delivery and is linked to various neurovascular disorders. In vitro BBB models provide a tool to investigate drug permeation across the BBB and the barrier's response to external injury events. Yet, existing models lack fidelity in replicating the BBB's complexity, hindering a comprehensive understanding of its functions. This study introduces a three-dimensional (3D) model using polyethylene glycol (PEG) hydrogels modified with biomimetic peptides that represent recognition sequences of key proteins in the brain. Hydrogels were functionalized with recognition sequences for laminin (IKVAV) and fibronectin peptides (RGD) and chemically cross-linked with matrix metalloprotease-sensitive peptides (MMPs) to mimic the extracellular matrix of the BBB. Astrocytes and endothelial cells were seeded within and on the surface of the hydrogels, respectively. The barrier integrity was assessed through different tests including transendothelial electrical resistance (TEER), the permeability of sodium fluorescence (Na-F), the permeability of Evan's blue bound to albumin (EBA), and the expression of zonula occluden-1 (ZO-1) in seeded endothelial cells. Hydrogels with a combination of RGD and IKVAV peptides displayed superior performance, exhibiting significantly higher TEER values (55.33 ± 1.47 Ω·cm2) at day 5 compared to other 2D controls including HAECs-monoculture and HAECs-cocultured with NHAs seeded on well inserts and 3D controls including RGD hydrogel and RGD-IKVAV monoculture with HAECs and RGD hydrogel cocultured with HAECs and NHAs. The designed 3D system resulted in the lowest Evan's blue permeability at 120 min (0.215 ± 0.055 μg/mL) compared to controls. ZO-1 expression was significantly higher and formed a relatively larger network in the functionalized hydrogel cocultured with astrocytes and endothelial cells compared to the controls. Thus, the designed 3D model effectively recapitulates the main BBB structure and function in vitro and is expected to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

Characteristics of Mitomycin C-Loaded Peptide Hydrogel In Vitro and Antiscarring Effects in Rat Ocular Injury Model.

Purpose: To investigate the characteristics of sustained drug release systems established by an arginine-glycine-aspartic acid (RGD) peptide hydrogel and mitomycin C (MMC) in vitro, and verify their antiscar effects in rat ocular injury model. Methods: Low, medium, and high loading doses of MMC were added to 5 mL 0.25%, 0.5%, and 1% wt RGD peptide hydrogel, respectively, to prepare 9 ratios of MMC-RGD systems. Drug release characteristics of the systems in phosphate-buffered saline solution were investigated by plotting the drug release curves and fitting them with mathematical models in OriginPro8.0 software. Appropriate ratios of MMC-RGD systems were selected as treatment in rat ocular injury model. Scar formation was observed by Masson staining and immunohistochemical staining with alpha-smooth muscle actin (α-SMA) and fibronectin (FN). Results: Nine ratios of MMC-RGD systems could release drug slowly. The maximum drug release proportions of all systems were >80%, and the time to maximum release proportions statistically prolonged with the increase of drug loading. Fitting with mathematical models indicated that the mechanisms of drug release were mainly Fick diffusion at early stage and Anomalous Transport at later stage. Systems of 1% wt RGD hydrogel were evaluated in animal experiments, which could inhibit hyperplasia of collagen and expression of α-SMA and FN. Conclusions: The RGD peptide hydrogel could be used as the carrier of MMC to establish sustained drug release system, which could inhibit scar formation after rat's ocular injury.

Amniotic membrane extract-enriched hydrogel augments the therapeutic effect of menstrual blood-derived stromal cells in a rat model of intrauterine adhesion

We previously demonstrated that transplantation of menstrual blood-derived stromal cells (MenSCs) is a safe and effective therapy for treating intrauterine adhesions (IUA). However, improving the colonization and therapeutic efficiency of MenSCs is still needed before full clinical application. Here, we established an amniotic membrane extract (AME)-enriched RGD hydrogel, and evaluated the therapeutic effect of this adjuvant combined with MenSCs transplantation in an IUA rat model. Our results indicated that AME promoted the proliferation and secretion of MenSCs in vitro, up-regulated the expression of apoptosis-suppressing gene BCL2 and down-regulated the expression of apoptosis-related genes Caspase-3 and Caspase-8. The AME-enriched hydrogel was biocompatible, and improved the survival of MenSCs in vitro and in vivo. It also promoted the retention of MenSCs in IUA uterus and augmented the effects of MenSCs on improving uterus morphology, endometrial proliferation, endometrial receptivity and fibrosis suppression. In addition, co-transplantation of MenSCs with AME-enriched hydrogel markedly down-regulated the expressions of inflammation-related genes IL10 and TGFβ while up-regulated the IL4/IFN-γ ratio in the IUA endometrium, and improved the expressions of cell proliferation-related antigen, gland-regeneration-related marker leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), angiogenesis-related marker platelet and endothelial cell adhesion molecule 1 (PECAM1), endometrial receptivity related genes ITGα5 and ITGβ3. Our study suggested that AME and MenSCs had a synergistic effect. Co-transplantation of MenSCs with AME-enriched hydrogel provided a promising approach for stem cell-based IUA treatment.

Recombinant Spider Silk Bioinks for Continuous Protein Release by Encapsulated Producer Cells.

Targeted therapies using biopharmaceuticals are of growing clinical importance in disease treatment. Currently, there are several limitations of protein-based therapeutics (biologicals), including suboptimal biodistribution, lack of stability, and systemic side effects. A promising approach to overcoming these limitations could be a therapeutic cell-loaded 3D construct consisting of a suitable matrix component that harbors producer cells continuously secreting the biological of interest. Here, the recombinant spider silk proteins eADF4(C16), eADF4(C16)-RGD, and eADF4(C16)-RGE have been processed together with HEK293 producer cells stably secreting the highly traceable reporter biological TNFR2-Fc-GpL, a fusion protein consisting of the extracellular domain of TNFR2, the Fc domain of human IgG1, and the luciferase of Gaussia princeps as a reporter domain. eADF4(C16) and eADF4(C16)-RGD hydrogels provide structural and mechanical support, promote HEK293 cell growth, and allow fusion protein production by the latter. Bioink-captured HEK293 producer cells continuously release functional TNFR2-Fc-GpL over 14 days. Thus, the combination of biocompatible, printable spider silk bioinks with drug-producing cells is promising for generating implantable 3D constructs for continuous targeted therapy.

HIF-1α overexpression in mesenchymal stem cell-derived exosome-encapsulated arginine-glycine-aspartate (RGD) hydrogels boost therapeutic efficacy of cardiac repair after myocardial infarction.

AimsNaturally secreted extracellular vesicles (EVs) play important roles in stem-mediated cardioprotection. This study aimed to investigate the cardioprotective function and underlying mechanisms of EVs derived from HIF-1α engineered mesenchymal stem cells (MSCs) in a rat model of AMI.Methods and resultsEVs isolated from HIF-1α engineered MSCs (HIF-1α-EVs) and control MSCs (NC-EVs) were prepared. In in vitro experiments, the EVs were incubated with cardiomyocytes and endothelial cells exposed to hypoxia and serum deprivation (H/SD); in in vivo experiments, the EVs were injected in the acutely infarcted hearts of Sprague-Dawley rats. Compared with NC-EVs, HIF-1α-EVs significantly inhibited the apoptosis of cardiomyocytes and enhanced angiogenesis of endothelial cells; meanwhile, HIF-1α-EVs also significantly shrunk fibrotic area and strengthened cardiac function in infarcted rats. After treatment with EVs/RGD-biotin hydrogels, we observed longer retention, higher stability in HIF-1α-EVs, and stronger cardiac function in the rats. Quantitative real-time PCR (qRT-PCR) displayed that miRNA-221–3p was highly expressed in HIF-1α-EVs. After miR-221–3p was inhibited in HIF-1α-EVs, the biological effects of HIF-1α EVs on apoptosis and angiogenesis were attenuated.ConclusionEVs released by MSCs with HIF-1α overexpression can promote the angiogenesis of endothelial cells and the apoptosis of cardiomyocytes via upregulating the expression of miR-221–3p. RGD hydrogels can enhance the therapeutic efficacy of HIF-1α engineered MSCs-derived EVs.

Enzymatically Cross-linked Hydrogels Based on Synthetic Poly(α-amino acid)s Functionalized with RGD Peptide for 3D Mesenchymal Stem Cell Culture.

Injectable hydrogel scaffolds combined with stem cell therapy represent a promising approach for minimally invasive surgical tissue repair. In this study, we developed and characterized a fully synthetic, biodegradable poly(N5-(2-hydroxyethyl)-l-glutamine)-based injectable hydrogel modified with integrin-binding arginine-glycine-aspartic acid (RGD) peptide (PHEG-Tyr-RGD). The biodegradable hydroxyphenyl polymer precursor derivative of PHEG-Tyr was enzymatically cross-linked to obtain injectable hydrogels with different physicochemical properties. The gelation time, gel yield, swelling behavior, and storage modulus of the PHEG-Tyr hydrogels were tuned by varying the concentrations of the PHEG-Tyr precursors and horseradish peroxidase as well as the nH2O2/nTyr ratio. The mechanical properties and gelation time of the PHEG-Tyr hydrogel were optimized for the encapsulation of rat mesenchymal stem cells (rMSCs). We focused on the 2D and 3D spreading and viability of rMSCs within the PHEG-Tyr-RGD hydrogels with different physicochemical microenvironments in vitro. Encapsulation of rMSCs shows long-term survival and exhibits cell-matrix and cell-cell interactions reflective of both the RGD concentration and hydrogel stiffness. The presented biomaterial represents a suitable biological microenvironment to guide 3D spreading and may act as a promising 3D artificial extracellular matrix for stem cell therapy.

Supramolecular Nanofibers Containing Arginine-Glycine-Aspartate (RGD) Peptides Boost Therapeutic Efficacy of Extracellular Vesicles in Kidney Repair.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSC-EVs) have been recognized as a promising cell-free therapy for acute kidney injury (AKI), which avoids safety concerns associated with direct cell engraftment. However, low stability and retention of MSC-EVs have limited their therapeutic efficacy. RGD (Arg-Gly-Asp) peptide binds strongly to integrins, which have been identified on the surface of MSC-EV membranes; yet RGD has not been applied to EV scaffolds to enhance and prolong bioavailability. Here, we developed RGD hydrogels, which we hypothesized could augment MSC-EV efficacy in the treatment of AKI models. In vivo tracking of the labeled EVs revealed that RGD hydrogels increased retention and stability of EVs. Integrin gene knockdown experiments confirmed that EV-hydrogel interaction was mediated by RGD-integrin binding. Upon intrarenal injection into mouse AKI models, EV-RGD hydrogels provided superior rescuing effects to renal function, attenuated histopathological damage, decreased tubular injury, and promoted cell proliferation in early phases of AKI. RGD hydrogels also augmented antifibrotic effects of MSC-EVs in chronic stages. Further analysis revealed that the presence of microRNA let-7a-5p in MSC-EVs served as the mechanism contributing to the reduced cell apoptosis and elevated cell autophagy in AKI. In conclusion, RGD hydrogels facilitated MSC-derived let-7a-5p-containing EVs, improving reparative potential against AKI. This study developed an RGD scaffold to increase the EV integrin-mediated loading and in turn improved therapeutic efficacy in renal repair; therefore this strategy shed light on MSC-EV application as a cell-free treatment for potentiated efficiency.

Hydrogel Culture Surface Stiffness Modulates Mesenchymal Stromal Cell Secretome and Alters Senescence.

Current cell culture surfaces used for the expansion and production of mesenchymal stromal cells (MSCs) are not optimized for the production of highly secretory and nonsenescent cells. In this study, we used poly (ethylene glycol) hydrogel substrates with tunable mechanical and biochemical properties to screen the effect of culture surfaces on pro-regenerative secretome by multiplex enzyme-linked immunosorbent assay, proliferation by PicoGreen DNA analysis, and senescence by senescence-associated β-galactosidase activity. We demonstrate that MSCs cultured on 30 kPa hydrogels, regardless of biochemical functionalization, broadly enhanced the secretion of immunomodulatory and regenerative factors versus stiffer 100 kPa or tissue culture plastic surfaces, but did not support robust proliferation. In contrast, culture on 100 kPa hydrogel surfaces promoted proliferation at a similar level and did not substantially alter the amount of secreted factors as compared with tissue culture plastic. Culture on integrin-engaging, cadherin-engaging, and hyaluronic acid-containing 30 kPa substrates enhanced MSC-conditioned media (CM) angiogenic activity in a human umbilical vein endothelial cell tube formation assay and human THP-1 monocyte chemoattraction in a transwell assay. However, 30 kPa substrate culture did not impact the myogenic activity of MSC CM in a C2C12 myoblast tube formation assay. Culture on selected 100 kPa surfaces enhanced CM angiogenic activity and monocyte chemotaxis, but not myogenic activity. Serial culture on 100 kPa RGD hydrogel surfaces significantly reduced senescence in MSCs versus tissue culture plastic, while maintaining the capacity of the cells to enhance their secretome in response to 30 kPa surfaces. Thus, hydrogel substrates that exhibit stiffness orders of magnitude lower than standard tissue culture plastic can serve as novel surfaces for the production of MSCs with an improved therapeutic secretory capacity and reduced senescence. Impact statement The success of mesenchymal stromal cell (MSC)-based therapies is dependent on the manufacture of a large number of cells with high therapeutic potency. Among the culture surfaces tested in this study, we demonstrate that substrate stiffness rather than biochemical functionalization predominantly guides changes in MSC proliferation and secretory capacity. We have identified substrate parameters to support MSC proliferation, enhance secretion of paracrine factors, and to reduce replicative senescence. By maximizing secretory capacity and reducing senescence through the choice of hydrogel culture materials, these findings have great potential to improve the large-scale production of therapeutic MSCs.

Pre-culture of mesenchymal stem cells within RGD-modified hyaluronic acid hydrogel improves their resilience to ischaemic conditions

The incorporation of the RGD peptide (arginine-glycine-aspartate) into biomaterials has been proposed to promote cell adhesion to the matrix, which can influence and control cell behaviour and function. While many studies have utilised RGD modified biomaterials for cell delivery, few have examined its effect under the condition of reduced oxygen and nutrients, as found at ischaemic injury sites. Here, we systematically examine the effect of RGD on hMSCs in hyaluronic acid (HA) hydrogel under standard and ischaemic culture conditions, to elucidate under what conditions RGD has beneficial effects over unmodified HA and its effectiveness in improving cell viability. Results demonstrate that under standard culture conditions, RGD significantly increased hMSC spreading and the release of vascular endothelial factor-1 (VEGF) and monocyte chemoattractant factor-1 (MCP-1), compared to unmodified HA hydrogel. As adhesion is known to influence cell survival, we hypothesised that cells in RGD hydrogels would exhibit increased cell viability under ischaemic culture conditions. However, results demonstrate that cell viability and protein release was comparable in both RGD modified and unmodified HA hydrogels. Confocal imaging revealed cellular morphology indicative of weak cell adhesion. Subsequent investigations found that RGD was could exert positive effects on encapsulated cells under ischaemic conditions but only if hMSCs were pre-cultured under standard conditions to allow strong adhesion to RGD before exposure. Together, these results provide novel insight into the value of RGD introduction and suggest that the adhesion of hMSCs to RGD prior to delivery could improve survival and function at ischaemic injury sites. Statement of significanceThe development of a biomaterial scaffold capable of maintaining cell viability while promoting cell function is a major research goal in the field of cardiac tissue engineering. This study confirms the suitability of a modified HA hydrogel whereby stem cells in the modified hydrogel showed significantly greater cell spreading and protein secretion compared to cells in the unmodified HA hydrogel. A pre-culture period allowing strong adhesion of the cells to the modified hydrogel was shown to improve cell survival under conditions that mimic the myocardium post-MI. This finding may have a significant impact on the use and timelines of modifications to improve stem cell survival in harsh environments like the injured heart.

Impact of RGD amount in dextran-based hydrogels for cell delivery

Dextran is one of the hydrophilic polymers that is used for hydrogel preparation. As any polysaccharide, it presents a high density of hydroxyl groups, which make possible several types of derivatization and crosslinking reactions. Furthermore, dextran is an excellent candidate for hydrogel fabrication with controlled cell/scaffold interactions as it is resistant to protein adsorption and cell adhesion. RGD peptide can be grafted to the dextran in order to promote selected cell adhesion and proliferation. Altogether, we have developed a novel strategy to graft the RGD peptide sequence to dextran-based hydrogel using divinyl sulfone as a linker. The resulting RGD functionalized dextran-based hydrogels were transparent, presented a smooth surface and were easy to handle. The impact of varying RGD peptide amounts, hydrogel porosity and topology upon human umbilical vein endothelial cell (HUVEC) adhesion, proliferation and infiltration was investigated. Our results demonstrated that 0.1% of RGD-modified dextran within the gel was sufficient to support HUVEC cells adhesion to the hydrogel surface. Sodium chloride was added (i) to the original hydrogel mix in order to form a macroporous structure presenting interconnected pores and (ii) to the hydrogel surface to create small orifices essential for cells migration inside the matrix.

Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide.

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during early porcine pregnancy and contains an Arg-Gly-Asp (RGD) peptide sequence that binds to cell surface integrins on the uterine endometrium and trophectoderm, promoting cell adhesion and migration. The aim of the present study was to evaluate the development of preimplantation porcine blastocysts encapsulated and cultured within alginate hydrogels either supplemented with SPP1 or conjugated with RGD. Blastocysts encapsulated within alginate hydrogels supplemented with SPP1 or conjugated with RGD had increased survival compared with non-encapsulated control blastocysts. In addition, the percentage of blastocysts encapsulated within RGD hydrogels that underwent morphological changes was greater than that of blastocysts encapsulated within standard alginate hydrogels or SPP1-supplemented hydrogels. Finally, only blastocysts encapsulated within RGD hydrogels had both increased expression of steroidogenic and immune responsiveness transcripts and increased 17β-oestradiol production, consistent with blastocysts undergoing elongation in vivo. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of blastocyst elongation.

Prevention of Medication-Related Osteonecrosis of the Jaw in a Rat Animal Model

Medication-related osteonecrosis of the jaw (MRONJ) still poses an enigma for clinicians and researchers alike, with a pathogenesis that is still shrouded in mystery. MRONJ is known to occur in patients taking oral or intravenous bisphosphonates following an invasive dental procedure of the bone yet, can also present rarely without any obvious inciting trauma. At present, there is no accepted preventative intervention of the the condition however, a variety of novel methods have been proposed ranging from the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) to the use of platelet rich fibrin (PRF) once MRONJ occurs. Currently, clinicians rely on prevention of the condition which revolves around identification of at-risk patients and minimization of exodontia or traumatic dental procedures involving bone after the commencement of bisphosphonate therapy. If MRONJ occurs, treatment is then dictated by the degree of bone exposure and spread to adjacent structures, through the use of antibiotics, antimicrobial mouthrinses or finally, surgery.1 This study evaluated the efficacy of a novel polyethylene glycol-heparin hydrogel2 (starPEG-heparin) incorporating arginylglycylaspartic acid (RGD) coupled with rhBMP-2 for the prevention of MRONJ development at the time of dentoalveolar surgery. Twenty-four (24) skeletally mature rats were divided into three equal groups (n=8) receiving two doses of intravenous zoledronic acid (0.1mg/Kg) at weeks two and five of the housing period prior to surgery. In all animals, the right first molar of the mandible was extracted and the gingiva sutured close using 6/0 prolene. Group 1 animals had the sockets left empty following extraction, while those in group 2 had starPEG-heparin/RGD hydrogel (15μL) without rhBMP-2 placed into the extraction socket. Animals in group 3 had starPEG-heparin /RGD hydrogel (15μL) with rhBMP-2 (5μg) placed in the extraction socket. Eight weeks after surgery specimens containing the right lower mandibular socket and contralateral equivalent were harvested and analyzed. Levels of c-terminal telopeptide (CTX) from serum taken periodically from the animals were evaluated using enzyme linked immunosorbent assays (ELISA). This demonstrated a linear downward trend in the bone turnover from week 1 to the final week of housing confirming the efficacy of the administered zoledronic acid. μCT analysis of samples showed a greater degree of bone formation in group 3 and the least bone formation in Group 1, as expected (P <0.05). Histological analysis of samples using H&E staining from group 1 displayed empty lacunae, or areas of karyorrhexis and pyknosis in the bone surrounding the extraction socket, consistent with MRONJ. Tartrate resistant acid phosphatase staining displayed an increase in osteoclastic action in groups 2 and 3 compared to group 1 (P <0.05). Although, increased angiogenesis was seen in groups 2 and 3 compared to group 1 throughout all the angiogenesis markers (anti-CD31, anti-CD34, anti-Von Willebrands Factor), these results were not significant. Scanning electron microscopic analysis of samples confirmed increased osteocyte density surrounding the extraction socket in groups 2 and 3 compared to group 1 (P <0.05). These results indicate that the use of a starPEG-heparin/RGD hydrogel combined with rhBMP-2 can stimulate increased osteoclastic action, localized angiogenesis and increased bone healing compared to both hydrogel-alone, or no intervention in a MRONJ rat animal model. Further work is required to evaluate if this novel intervention is translatable to an animal model with bone biology more similar to the human species.

Integrin-specific hydrogels functionalized with VEGF for vascularization and bone regeneration of critical-size bone defects.

Vascularization of bone defects is considered a crucial component to the successful regeneration of large bone defects. Although vascular endothelial growth factor (VEGF) has been delivered to critical-size bone defect models to augment blood vessel infiltration into the defect area, its potential to increase bone repair remains ambiguous. In this study, we investigated whether integrin-specific biomaterials modulate the effects of VEGF on bone regeneration. We engineered protease-degradable, VEGF-loaded poly(ethylene glycol) (PEG) hydrogels functionalized with either a triple-helical, α2 β1 integrin-specific peptide GGYGGGP(GPP)5 GFOGER(GPP)5 GPC (GFOGER) or an αv β3 integrin-targeting peptide GRGDSPC (RGD). Covalent incorporation of VEGF into the PEG hydrogel allowed for protease degradation-dependent release of the protein while maintaining VEGF bioactivity. When applied to critical-size segmental defects in the murine radius, GFOGER-functionalized VEGF-free hydrogels exhibited significantly increased vascular volume and density and resulted in a larger number of thicker blood vessels compared to RGD-functionalized VEGF-free hydrogels. VEGF-loaded RGD hydrogels increased vascularization compared to VEGF-free RGD hydrogels, but the levels of vascularization for these VEGF-containing RGD hydrogels were similar to those of VEGF-free GFOGER hydrogels. VEGF transiently increased bone regeneration in RGD hydrogels but had no effect at later time points. In GFOGER hydrogels, VEGF did not show an effect on bone regeneration. However, VEGF-free GFOGER hydrogels resulted in increased bone regeneration compared to VEGF-free RGD hydrogels. These findings demonstrate the importance of integrin-specificity in engineering constructs for vascularization and associated bone regeneration.

Integrin-specific hydrogels for increased vascularization and bone regeneration of critically-sized bone defects

Event Abstract Back to Event Integrin-specific hydrogels for increased vascularization and bone regeneration of critically-sized bone defects Jose Garcia1, 2 and Andres J. Garcia1, 2 1 Georgia Institute of Technology, Woodruff Scool of Mechanical Engineering, United States 2 Georgia Institute of Technology, Institute of Bioengineering and Biosciences, United States Introduction: Non-healing bone defects and fractures represent a serious clinical problem with over 600,000 bone replacement procedures performed each year. The lack of functional vasculature following trauma or resection of bone has emerged as a major limitation that prevents full regeneration of the defect[1]. While numerous scaffolds have been engineered to increase regeneration of the vascular network, the majority use growth factors at supraphysiological levels which increase the cost of potential therapies. Engineering scaffolds that inherently promote revascularization based on the material’s interaction with surrounding tissue can be an effective strategy for healing the defect. In this study, we investigated how integrin specificity in the biomaterial influences vascularization of defects with the hypothesis that integrin specificity will enhance vascularization. Methods: Hydrogel Synthesis: Integrin-specific hydrogels were engineered by functionalizing 4-arm PEG-maleimide macromers with either the α2β1-specific ‘GFOGER’ peptide[2] or the αvβ3-targeting ‘RGD’ peptide followed by functionalization with vascular endothelial growth factor (VEGF) and cross-linked with a protease-degradable peptide. Vasculogenic protein analysis: Human mesenchymal stem cells (hMSCs) were encapsulated within integrin-specific hydrogels and cultured in osteogenic differentiation media for 7 days. Conditioned media was then assayed for VEGF by ELISA. Critically-sized murine radial defect: For in vivo studies, hydrogels were implanted within a 2.5 mm long critically-sized murine radial bone defect. At 8 weeks, animals were perfused with a radiopaque polymer (Microfil) and µCT analysis performed for vasculature. Bone formation was also analyzed by µCT at 4 and 8 weeks. Results and Discussion: hMSCs cultured within GFOGER-functionalized hydrogels in osteogenic differentiation media exhibited increased secretion of VEGF compared to hMSCs within RGD hydrogels (Fig. 1). In vivo, GFOGER-functionalized VEGF-free hydrogels exhibited significantly increased vascular volume (Fig. 2A), density (Fig. 2B) and resulted in a larger number (counts) of thicker blood vessels compared to RGD-functionalized VEGF-free hydrogels (Fig. 2C-D). The levels of vasculogenesis for VEGF-free GFOGER hydrogels were similar to those of RGD hydrogels delivering 50 ng of VEGF. VEGF-free GFOGER hydrogels also exhibited significantly increased bone volume at 4 and 8 weeks compared to VEGF-free RGD hydrogels (0.028 ± 0.008 mm3 vs 0.004 ± 0.001 mm3 and 0.06 ± 0.02 mm3 vs 0.01 ± 0.006 mm3 for 4 and 8 weeks respectively, mean ±SEM, p<0.05) Conclusion: Whereas growth factors are routinely used to increase vascular regeneration in vivo, their high cost as well as their detrimental effects at supraphysiological levels demands exploration of more viable strategies. Integrin-specificity is a crucial aspect of vascularization and represents a useful tool in engineering constructs for this purpose. Cellular interaction with α2β1-specific hydrogels caused increased secretion of VEGF which correlated with increased vascularization and bone regeneration of a bone defect in vivo. Future biomaterial should consider integrin specificity to reduce or even avoid the use of growth factors. Dr. Nick Willet; Amy Clark; Funding provided by NIH grants R01 AR062368 and R01 AR062920.

Integrin specificity as a novel strategy for enhancing transplanted stem cell survival and tissue repair in vivo

Event Abstract Back to Event Integrin specificity as a novel strategy for enhancing transplanted stem cell survival and tissue repair in vivo Amy Clark1, 2* and Andres J. Garcia1, 2 1 Georgia Institute of Technology, Woodruff School of Mechanical Engineering, United States 2 Georgia Institute of Technology, Petit Institute for Bioengineering and Bioscience, United States Introduction: Despite the promising clinical results for the use of human mesenchymal stem cells (hMSC) in musculoskeletal defect treatment, inadequate control over cell survival, engraftment and fate limits the success of this cell-based therapy. Integrin-mediated cell adhesion plays a central role in tissue formation, maintenance, and repair by providing anchorage forces and triggering signals that regulate cell function. We hypothesize biomaterials presenting integrin-specific adhesive motifs will direct hMSC signaling and specification. The objective of this project is to engineer bioartificial hydrogels presenting integrin-specific ligands as biomimetic cell delivery vehicles for enhanced in vivo engraftment and function – an innovative strategy as it focuses on engineering specificity to integrin receptors to promote survival and cell-based repair without the use of exogenous growth factors. Methods: Early passage hMSC (<4) were transduced to constitutively express firefly luciferase (hMSCFLuc). 4-arm PEG-maleimide was functionalized with one of three ligands: α2β1-binding collagen I-mimetic GFOGER, αvβ3-binding RGD, or non-adhesive RDG peptides and cross-linked with a protease-degradable peptide. 2.5 mm defects were created in the radii of 8-9 week old male NOD scid gamma mice and treated with cell-laden hydrogels containing 15k hMSC. Bioluminescence was monitored using an In Vivo Imaging System (PerkinElmer). New bone formation was evaluated using an in vivo microcomputer tomography scanner (Scanco Medical). Gene expression was evaluated after 1 week in vivo. Total RNA was extracted from cells in the defect space and standardized across all samples. 96 total genes were screened using Fluidigm gene chip technology. Results: Bioluminescence of transplanted hMSCFLuc was monitored after transplantation (Fig 1A). The significantly higher bioluminescent signal in GFOGER/hMSCFLuc-treated defects suggests GFOGER hydrogels support transplanted cell survival and proliferation in vivo whereas the bioluminescent signals for hMSCFLuc delivered in a RGD or RDG hydrogel remained low. GFOGER/hMSC-treated defects exhibited significantly higher bone volume at weeks 4 and 8 compared to RGD and RDG with no statistical difference between groups receiving hMSCFLuc and hMSC (Fig 1B). Differences in gene expression for approximately 60 genes were observed after 1 week in vivo, with statistically higher expression of vascularization and inflammation genes in GFOGER/hMSC treated defects (Fig 2). Conclusions: GFOGER-functionalized PEG hydrogels support enhanced bone formation and hMSC viability in vivo compared to RGD and RDG, possibly via enhanced vascularization These studies are ongoing for immunohistochemical, cytokine, and defect vascularization analyses. This work highlights integrin-specificity as an important consideration in the design of cell delivery vehicles for engraftment and tissue repair. NIH grants R01 AR062368 and R01 AR062920

Modulation of the stemness and osteogenic differentiation of human mesenchymal stem cells by controlling RGD concentrations of poly(carboxybetaine) hydrogel.

In vitro modulation of the differentiation status of mesenchymal stem cells (MSCs) is important for their application to regenerative medicine. We suggested that the morphology and differentiation states of MSCs could be modulated by controlling the cell affinity of a substrate. The objective of this study was to investigate the effects of surface bio-adhesive signals on self-renewal and osteogenic differentiation of MSCs using a low-fouling platform. Cell-resistant poly(carboxybetaine) hydrogel was conjugated with 5 μM or 5 mM of cell-adhesive arginine-glycine-aspartic acid (RGD) peptides in order to control the cells' affinity to the substrate. Human mesenchymal stem cells (hMSCs) were cultured on the RGD-modified poly(carboxybetaine) hydrogel and then the cells' states of stemness and osteogenic differentiation were evaluated using reverse-transcriptase polymerase chain reaction. The hMSCs formed three-dimensional spheroids on the 5 μM RGD substrate, while cells on the 5 mM RGD substrate exhibited spreading morphology. Furthermore, cells on the 5 μM RGD hydrogel maintained a better stemness phenotype, while the hMSCs on the 5 mM RGD hydrogel proliferated faster and underwent osteogenic differentiation. In conclusion, the stemness of hMSCs was best maintained on a low RGD surface, while osteogenic differentiation of hMSCs was enhanced on a high RGD surface.

Behavior of encapsulated MG-63 cells in RGD and gelatine-modified alginate hydrogels.

Achieving cell spreading and proliferation inside hydrogels that are compatible with microencapsulation technology represents a major challenge for tissue engineering scaffolding and for the development of three-dimensional cell culture models. In this study, microcapsules of 650-900 μm in diameter were fabricated from oxidized alginate covalently cross-linked with gelatine (AlGel). Schiff's base bond formed in AlGel, detected by Fourier transform infrared spectroscopy, which confirmed the cross-linking of oxidized alginate with gelatine. Biological properties of alginate based hydrogels were studied by comparing the viability and morphology of MG-63 osteosarcoma cells encapsulated in gelatine and RGD-modified alginate. We hypothesized that the presence of gelatine and RGD will support cell adhesion and spreading inside the microcapsules and finally, also vascular endothelial growth factor (VEGF) secretion. After 4 days of incubation, cells formed extensive cortical protrusions and after 2 weeks they proliferated, migrated, and formed cellular networks through the AlGel material. In contrast, cells encapsulated in pure alginate and in RGD-modified alginate formed spherical aggregates with limited cell mobility and VEGF secretion. Metabolic activity was doubled after 5 days of incubation, making AlGel a promising material for cell encapsulation.

Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle

Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.

Genetically Engineered Myoblast Sheet for Therapeutic Angiogenesis

Peripheral arterial disease is a common manifestation of systemic atherosclerosis, which results in more serious consequences of ischemic events in peripheral tissues such as the lower extremities. Cell therapy has been tested as a treatment for peripheral ischemia that functions by inducing angiogenesis in the ischemic region. However, the poor survival and engraftment of transplanted cells limit the efficacy of cell therapy. In order to overcome such challenges, we applied genetically engineered cell sheets using a cell-interactive and thermosensitive hydrogel and nonviral polymer nanoparticles. C2C12 myoblast sheets were formed on Tetronic-tyramine (Tet-TA)-RGD hydrogel prepared through a highly efficient and noncytotoxic enzymatic reaction. The myoblast sheets were then transfected with vascular endothelial growth factor (VEGF) plasmids using poly(β-amino ester) nanoparticles to increase the angiogenic potential of the sheets. The transfection increased the VEGF expression and secretion from the C2C12 sheets. The enhanced angiogenic effect of the VEGF-transfected C2C12 sheets was confirmed using an in vitro capillary formation assay. More importantly, the transplantation of the VEGF-transfected C2C12 sheets promoted the formation of capillaries and arterioles in ischemic muscles, attenuated the muscle necrosis and fibrosis progressed by ischemia, and eventually prevented ischemic limb loss. In conclusion, the combination of cell sheet engineering and genetic modification can provide more effective treatment for therapeutic angiogenesis.

Alginate hydrogels containing cell‐interactive beads for bone formation

Alginate hydrogels containing cell-instructive cues are the subject of intense interest for their use as cell carriers in bone tissue engineering. Peptides and proteins are chemically grafted onto these hydrophilic materials to facilitate adhesion and direct phenotype of entrapped cells. However, the presentation of a single or small number of peptides does not represent the complexity of the native extracellular matrix (ECM) of bony tissues. Mesenchymal stem cells (MSCs) secrete ECM that can be harvested and deposited on various substrata to promote osteogenic differentiation. In this study, we hypothesized that the presentation of engineered cell-secreted ECM on microbeads suspended in alginate hydrogels would promote cell adhesion and enhance osteogenic differentiation of undifferentiated MSCs without chemical incorporation of cell-adhesive peptides. Human MSCs entrapped in alginate hydrogels loaded with ECM-coated beads showed increased interaction with beads, when compared with cells suspended in hydrogels containing uncoated blank (BLK) beads. MSCs entrapped in ECM gels exhibited increased alkaline phosphatase (ALP) activity and expression of osteogenic genes in vitro compared with hydrogels modified with arginine-glycine-aspartic acid (RGD)-containing peptides. Transplantation of MSCs into an ectopic site resulted in significant increases in blood vessel density for ECM hydrogels when compared with the BLK or RGD gels. Furthermore, we observed comparable levels of bone formation at 6 wk with ECM and RGD hydrogels. These findings demonstrate that engineered ECM can be deployed in a minimally invasive manner to direct the formation of bony tissue. This strategy may provide an alternative to the engraftment of proteins or peptides onto the polymer backbone of hydrogels for directing cellular behavior.