Development of Human-Derived Photocrosslinkable Gelatin Hydrogels for Tissue Engineering.

Hydrogels are often used as biomimetic matrices for tissue regeneration. The source of the hydrogel is of utmost importance, as it affects the physicochemical characteristics and must be carefully selected to stimulate specific cell behaviors. Naturally derived polymeric biomaterials have inherent biological moieties, such as cell binding and protease cleavage sites, and thus can provide a suitable microenvironment for cells. Human-derived matrices can mitigate potential risks associated with the immune response and disease transmission from animal-derived biomaterials. In this article, we developed glycidyl methacrylate-modified human-derived gelatin (hGelGMA) hydrogels for use in tissue engineering applications. By adjusting the glycidyl methacrylate concentration in the reaction mixture, we synthesized hGelGMA with low, medium, and high degrees of modification referred to as hGelGMA-L, hGelGMA-M, and hGelGMA-H, respectively. The amount of polymeric networks in the hydrogels was increased proportionally with the degree of modification. This change has resulted in a decreasing trend in pore size, porosity, and consequent swelling ratio. Similarly, increasing the polymer concentration also exhibited slower enzymatic degradation. On the other hand, increasing the polymer concentration led to an improvement in mechanical properties, where the compressive moduli of hGelGMA-L, hGelGMA-M, and hGelGMA-H hydrogels have changed at 2.9 ± 1.0, 13.7 ± 0.9, and 26.4 ± 2.5 kPa, respectively. The cytocompatibility of hGelGMA was assessed by 3D encapsulation of human-derived cells, including human dermal fibroblasts (HDFs) and human mesenchymal stem cells (hMSCs), in vitro. Regardless of the degree of glycidyl methacrylate modification, the hGelGMA hydrogels preserved the viability of encapsulated cells and supported their growth and proliferation. HDF cells showed a higher metabolic activity in hGelGMA-H, while MSCs exhibited an increased metabolic activity when they were encapsulated in hGelGMA-M or hGelGMA-H. These results showed that photocrosslinkable human-derived gelatin-based hydrogels can be synthesized and their physical properties can be distinctly fine-tuned to different extents as a function of their degrees of modification depending on the needs of the target tissue. Due to its promising physical and biological properties, it is anticipated that hGelGMA can be utilized in a wide spectrum of tissue engineering applications.

Synthesis and characterization of photocrosslinkable albumin-based hydrogels for biomedical applications.

Protein-based biomaterials are widely used to generate three-dimensional (3D) scaffolds for tissue regeneration as well as compact delivery systems for drugs, genes, and peptides. Specifically, albumin-based biomaterials are of particular interest for their ability to facilitate controlled delivery of drugs and other therapeutic agents. These hydrogels possess non-toxic and non-immunogenic properties that are desired in tissue engineering scaffolds. This work employs a rapid ultraviolet (UV) light induced crosslinking to fabricate bovine serum albumin (BSA) hydrogels. Using four different conditions, the BSA hydrogel properties were modulated based on the extent of glycidyl methacrylate modification in each polymer. The highly tunable mechanical behavior of the material was determined through compression tests which yielded a range of material strengths from 4.4 ± 1.5 to 122 ± 7.4 kPa. Pore size measurements also varied from 7.7 ± 1.7 to 23.5 ± 6.6 µm in the photocrosslinked gels. The physical properties of materials such as swelling and degradation were also characterized. In further evaluation, 3D scaffolds were used in cell encapsulation and in vivo implantation studies. The biocompatibility and degradability of the material demonstrated effective integration with the native tissue environment. These modifiable chemical and mechanical properties allow BSA hydrogels to be fine-tuned to a plethora of biomedical applications including regenerative medicine, in vitro cancer study models, and wound healing approaches.

3D Printed Eggshell Microparticle-Laden Thermoplastic Scaffolds for Bone Tissue Engineering.

Three-dimensional (3D) printing, an additive manufacturing technique, is increasingly used in the field of tissue engineering. The ability to create complex structures with high precision makes the 3D printing of this material a preferred method for constructing personalized and functional materials. However, the challenge lies in developing affordable and accessible materials with the desired physiochemical and biological properties. In this study, we used eggshell microparticles (ESPs), an example of bioceramic and unconventional biomaterials, to reinforce thermoplastic poly(ε-caprolactone) (PCL) scaffolds via extrusion-based 3D printing. The goal was to conceive a sustainable, affordable, and unique personalized medicine approach. The scaffolds were fabricated with varying concentrations of eggshells, ranging from 0 to 50% (w/w) in the PCL scaffolds. To assess the physicochemical properties, we employed scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, and X-ray diffraction analysis. Mechanical properties were evaluated through compression testing, and degradation kinetics were studied through accelerated degradation with the remaining mass ranging between 89.4 and 28.3%. In vitro, we evaluated the characteristics of the scaffolds using the MC3T3-E1 preosteoblasts over a 14 day period. In vitro characterization involved the use of the Alamar blue assay, confocal imaging, and real-time quantitative polymerase chain reaction. The results of this study demonstrate the potential of 3D printed biocomposite scaffolds, consisting of thermoplastic PCL reinforced with ESPs, as a promising alternative for bone-graft applications.

Stem Cells in Bone Tissue Engineering: Progress, Promises and Challenges.

Bone defects from accidents, congenital conditions, and age-related diseases significantly impact quality of life. Recent advancements in bone tissue engineering (TE) involve biomaterial scaffolds, patient-derived cells, and bioactive agents, enabling functional bone regeneration. Stem cells, obtained from numerous sources including umbilical cord blood, adipose tissue, bone marrow, and dental pulp, hold immense potential in bone TE. Induced pluripotent stem cells and genetically modified stem cells can also be used. Proper manipulation of physical, chemical, and biological stimulation is crucial for their proliferation, maintenance, and differentiation. Stem cells contribute to osteogenesis, osteoinduction, angiogenesis, and mineralization, essential for bone regeneration. This review provides an overview of the latest developments in stem cell-based TE for repairing and regenerating defective bones.

Synthesis and characterization of hybrid hyaluronic acid-gelatin hydrogels.

Biomimetic hybrid hydrogels have generated broad interest in tissue engineering and regenerative medicine. Hyaluronic acid (HA) and gelatin (hydrolyzed collagen) are naturally derived polymers and biodegradable under physiological conditions. Moreover, collagen and HA are major components of the extracellular matrix (ECM) in most of the tissues (e.g., cardiovascular, cartilage, neural). When used as a hybrid material, HA-gelatin hydrogels may enable mimicking the ECM of native tissues. Although HA-gelatin hybrid hydrogels are promising biomimetic substrates, their material properties have not been thoroughly characterized in the literature. Herein, we generated hybrid hydrogels with tunable physical and biological properties by using different concentrations of HA and gelatin. The physical properties of the fabricated hydrogels including swelling ratio, degradation, and mechanical properties were investigated. In addition, in vitro cellular responses in both two and three-dimensional culture conditions were assessed. It was found that the addition of gelatin methacrylate (GelMA) into HA methacrylate (HAMA) promoted cell spreading in the hybrid hydogels. Moreover, the hybrid hydrogels showed significantly improved mechanical properties compared to their single component analogs. The HAMA-GelMA hydrogels exhibited remarkable tunability behavior and may be useful for cardiovascular tissue engineering applications.

Oxygen-Generating Scaffolds for Cardiac Tissue Engineering Applications.

Homogeneous vascularization of implanted tissue constructs can extend to 5 weeks, during which cell death can occur due to inadequate availability of oxygen. Researchers are engineering biomaterials that generate and release oxygen in a regulated manner, in an effort to overcome this hurdle. A main limitation of the existing oxygen-generating biomaterials is the uncontrolled release of oxygen, which is ultimately detrimental to the cells. This study demonstrates the incorporation of calcium peroxide (CaO2) within a hydrophobic polymer, polycaprolactone (PCL), to yield composite scaffolds with controlled oxygen release kinetics sustained over 5 weeks. Oxygen-generating microparticles coencapsulated with cardiomyocytes in a gelatin-based hydrogel matrix can serve as model systems for cardiac tissue engineering. Specifically, the results reveal that the oxygen-generating microspheres significantly improve the scaffold mechanical strength ranging from 5 kPa to 35 kPa, have an average scaffold pore size of 50-100 µm, swelling ratios of 33.3-29.8%, and degradation with 10-49% remaining mass at the end of a 48 h accelerated enzymatic degradation. The oxygen-generating scaffolds demonstrate improvement in cell viability, proliferation, and metabolic activity compared to the negative control group when cultured under hypoxia. Additionally, the optimized oxygen-generating constructs display no cytotoxicity or apoptosis. These oxygen-generating scaffolds can possibly assist the in vivo translation of cardiac tissue constructs.

Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions.

BACKGROUND: Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell-cell interactions with microscale resolution. SCOPE OF THE REVIEW: We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell-cell interactions in the resulting biological function of the tissues. MAJOR CONCLUSIONS: Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell-cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell-cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell-matrix interactions. GENERAL SIGNIFICANCE: Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

Responsive micromolds for sequential patterning of hydrogel microstructures.

Microscale hydrogels have been shown to be beneficial for various applications such as tissue engineering and drug delivery. A key aspect in these applications is the spatial organization of biological entities or chemical compounds within hydrogel microstructures. For this purpose, sequentially patterned microgels can be used to spatially organize either living materials to mimic biological complexity or multiple chemicals to design functional microparticles for drug delivery. Photolithographic methods are the most common way to pattern microscale hydrogels but are limited to photocrosslinkable polymers. So far, conventional micromolding approaches use static molds to fabricate structures, limiting the resulting shapes that can be generated. Herein, we describe a dynamic micromolding technique to fabricate sequentially patterned hydrogel microstructures by exploiting the thermoresponsiveness of poly(N-isopropylacrylamide)-based micromolds. These responsive micromolds exhibited shape changes under temperature variations, facilitating the sequential molding of microgels at two different temperatures. We fabricated multicompartmental striped, cylindrical, and cubic microgels that encapsulated fluorescent polymer microspheres or different cell types. These responsive micromolds can be used to immobilize living materials or chemicals into sequentially patterned hydrogel microstructures which may potentially be useful for a range of applications at the interface of chemistry, materials science and engineering, and biology.

Eggshell Microparticle Reinforced Scaffolds for Regeneration of Critical Sized Cranial Defects.

Scaffold-based approaches for bone regeneration have been studied using a wide range of biomaterials as reinforcing agents to improve the mechanical strength and bioactivity of the 3D constructs. Eggshells are sustainable and inexpensive materials with unique biological and chemical properties to support bone differentiation. The incorporation of eggshell particles within hydrogels yields highly osteoinductive and osteoconductive scaffolds. This study reveals the effects of microparticles of whole eggshells, eggshells without a membrane, and a pristine eggshell membrane on osteogenic differentiation in protein-derived hydrogels. The in vitro studies showed that gels reinforced with eggshells with and without a membrane demonstrated comparable cellular proliferation, osteogenic gene expression, and osteogenic differentiation. Subsequently, in vivo studies were performed to implant eggshell microparticle-reinforced composite hydrogel scaffolds into critical-sized cranial defects in Sprague Dawley (SD) rats for up to 12 weeks to study bone regeneration. The in vivo results showed that the eggshell microparticle-based scaffolds supported an average bone volume of 60 mm3 and a bone density of 2000 HU 12 weeks post implantation. Furthermore, histological analyses of the explanted scaffolds showed that the eggshell microparticle-reinforced scaffolds permitted tissue infiltration and induced bone tissue formation over 12 weeks. The histology staining also indicated that these scaffolds induced significantly higher bone regeneration at 6 and 12 weeks as compared to the blank (no scaffold) and pristine gel scaffolds. The eggshell microparticle-reinforced scaffolds also supported significantly higher bone formation, remodeling, and vascularization over 6 and 12 weeks as confirmed by immunohistochemistry analysis. Collectively, our results indicated that eggshell microparticle-reinforced scaffolds facilitated significant bone regeneration in critical-sized cranial defects.

Engineering calcium peroxide based oxygen generating scaffolds for tissue survival.

Oxygen supply is essential for the long-term viability and function of tissue engineered constructs in vitro and in vivo. The integration with the host blood supply as the primary source of oxygen to cells requires 4 to 5 weeks in vivo and involves neovascularization stages to support the delivery of oxygenated blood to cells. Consequently, three-dimensional (3D) encapsulated cells during this process are prone to oxygen deprivation, cellular dysfunction, damage, and hypoxia-induced necrosis. Here we demonstrate the use of calcium peroxide (CaO2) and polycaprolactone (PCL), as part of an emerging paradigm of oxygen-generating scaffolds that substitute the host oxygen supply via hydrolytic degradation. The 35-day in vitro study showed predictable oxygen release kinetics that achieved 5% to 29% dissolved oxygen with increasing CaO2 loading. As a biomaterial, the iterations of 0 mg, 40 mg, and 60 mg of CaO2 loaded scaffolds yielded modular mechanical behaviors, ranging from 5-20 kPa in compressive strength. The other controlled physiochemical features included swelling capacities of 22-33% and enzymatic degradation rates of 0.8% to 60% remaining mass. The 3D-encapsulation experiments of NIH/3T3 fibroblasts, L6 rat myoblasts, and primary cardiac fibroblasts in these scaffolds showed enhanced cell survival, proliferation, and function under hypoxia. During continuous oxygen release, the scaffolds maintained a stable tissue culture system between pH 8 to 9. The broad basis of this work supports prospects in the expansion of robust and clinically translatable tissue constructs.

3D Printing of Micro- and Nanoscale Bone Substitutes: A Review on Technical and Translational Perspectives.

Recent developments in three-dimensional (3D) printing technology offer immense potential in fabricating scaffolds and implants for various biomedical applications, especially for bone repair and regeneration. As the availability of autologous bone sources and commercial products is limited and surgical methods do not help in complete regeneration, it is necessary to develop alternative approaches for repairing large segmental bone defects. The 3D printing technology can effectively integrate different types of living cells within a 3D construct made up of conventional micro- or nanoscale biomaterials to create an artificial bone graft capable of regenerating the damaged tissues. This article reviews the developments and applications of 3D printing in bone tissue engineering and highlights the numerous conventional biomaterials and nanomaterials that have been used in the production of 3D-printed scaffolds. A comprehensive overview of the 3D printing methods such as stereolithography (SLA), selective laser sintering (SLS), fused deposition modeling (FDM), and ink-jet 3D printing, and their technical and clinical applications in bone repair and regeneration has been provided. The review is expected to be useful for readers to gain an insight into the state-of-the-art of 3D printing of bone substitutes and their translational perspectives.

Unconventional Tissue Engineering Materials in Disguise.

Tissue engineering faces a recurring challenge in the transformation of biomaterials into 3D constructs that mimic the biological, chemical, and mechanical features of native tissues. Some of the conventional approaches can be sophisticated and involve extensive material processing and high-cost fabrication procedures. Despite tremendous strides in biomaterials discovery and characterization, the functional and manufacturing limitations have led to the innovation of novel biomimetic techniques that borrow from nature, human-made commodities, and other parts of life to overcome the challenges in tissue engineering and regenerative medicine. This review explores engineering strategies that involve unusual materials for improved functionality, scalability, sustainability, and cost-efficiency. The biomaterials discussed are globally accessible resources and can serve across a wide spectrum of biomedical research areas.

Hydroxyapatite-Incorporated Composite Gels Improve Mechanical Properties and Bioactivity of Bone Scaffolds.

Reinforcing polymeric scaffolds with micro/nanoparticles improve their mechanical properties and render them bioactive. In this study, hydroxyapatite (HA) is incorporated into 5% (w/v) gelatin methacrylate (GelMA) hydrogels at 1, 5, and 20 mg mL-1 concentrations. The material properties of these composite gels are characterized through swelling, degradation, and compression tests. Using 3D cell encapsulation, the cytocompatibility and osteogenic differentiation of preosteoblasts are evaluated to assess the biological properties of the composite scaffolds. The in vitro assays demonstrate increasing cell proliferation and metabolic activity over the course of 14 d in culture. Furthermore, the scaffolds support osteogenic differentiation of the microencapsulated preosteoblasts. For the in vivo study, the composite scaffolds are subcutaneously implanted in rats for 14 d. The histological staining of the explanted in vivo samples exhibits the functional advantages of the scaffold's biocompatibility, biodegradability, and integration into the existing host tissue. This work demonstrates the enhanced mechanical and biological performance of HA-gelatin composite hydrogels for bone tissue engineering applications.

Eggshell particle-reinforced hydrogels for bone tissue engineering: an orthogonal approach.

Hydrogel-based biomimetic scaffolds have generated broad interest due to their tunable physical, chemical, and biological properties for bone tissue engineering applications. We fabricated eggshell microparticle (ESP) reinforced gelatin-based hydrogels to obtain mechanically stable and biologically active three-dimensional (3D) constructs that can differentiate pre-mature cells into osteoblasts. Physical properties including swelling ratio, degradation, and mechanical properties of the composite hydrogels were investigated. Pre-osteoblasts were encapsulated within the ESP-reinforced hydrogels to study their differentiation and evaluate mineral deposition by these cells. The ESP-reinforced gels were then subcutaneously implanted in a rat model to determine their biocompatibility and degradation behaviors. The composite hydrogels have shown outstanding tunability in physical and biological properties holding substantial promise for engineering mineralized tissues (e.g. bone, cartilage, tooth, and tendon). These 3D scaffolds enabled the differentiation of pre-osteoblasts without the use of specialized osteogenic growth medium. The ESP-reinforced gels exhibited significant enhancement in mineralization by pre-osteoblasts. These behaviors are positively correlated with increasing concentrations of ESP. Findings suggest that our novel composite hydrogel exhibits superior mechanical properties and indicates a favorable in vivo response by subcutaneous implantation in a rat model.

High-throughput approaches for screening and analysis of cell behaviors.

The rapid development of new biomaterials and techniques to modify them challenge our capability to characterize them using conventional methods. In response, numerous high-throughput (HT) strategies are being developed to analyze biomaterials and their interactions with cells using combinatorial approaches. Moreover, these systematic analyses have the power to uncover effects of delivered soluble bioactive molecules on cell responses. In this review, we describe the recent developments in HT approaches that help identify cellular microenvironments affecting cell behaviors and highlight HT screening of biochemical libraries for gene delivery, drug discovery, and toxicological studies. We also discuss HT techniques for the analyses of cell secreted biomolecules and provide perspectives on the future utility of HT approaches in biomedical engineering.

Engineered Paper-Based Cell Culture Platforms.

Paper is used in various applications in biomedical research including diagnostics, separations, and cell cultures. Paper can be conveniently engineered due to its tunable and flexible nature, and is amenable to high-throughput sample preparation and analysis. Paper-based platforms are used to culture primary cells, tumor cells, patient biopsies, stem cells, fibroblasts, osteoblasts, immune cells, bacteria, fungi, and plant cells. These platforms are compatible with standard analytical assays that are typically used to monitor cell behavior. Due to its thickness and porous nature, there are no mass transport limitations to/from the cells in paper scaffolds. It is possible to pattern paper in different scales (micrometer to centimeter), generate modular configurations in 3D, fabricate multicellular and compartmentalized tissue mimetics for clinical applications, and recover cells from the scaffolds for further analysis. 3D paper constructs can provide physiologically relevant tissue models for personalized medicine. Layer-by layer strategies to assemble tissue-like structures from low-cost and biocompatible paper-based materials offer unique opportunities that include understanding fundamental biology, developing disease models, and assembling different tissues for organ-on-paper applications. Paper-based platforms can also be used for origami-inspired tissue engineering. This work provides an overview of recent progress in engineered paper-based biomaterials and platforms to culture and analyze cells.

Gradient static-strain stimulation in a microfluidic chip for 3D cellular alignment.

Cell alignment is a critical factor to govern cellular behavior and function for various tissue engineering applications ranging from cardiac to neural regeneration. In addition to physical geometry, strain is a crucial parameter to manipulate cellular alignment for functional tissue formation. In this paper, we introduce a simple approach to generate a range of gradient static strains without external mechanical control for the stimulation of cellular behavior within 3D biomimetic hydrogel microenvironments. A glass-supported microfluidic chip with a convex flexible polydimethylsiloxane (PDMS) membrane on the top was employed for loading the cells suspended in a prepolymer solution. Following UV crosslinking through a photomask with a concentric circular pattern, the cell-laden hydrogels were formed in a height gradient from the center (maximum) to the boundary (minimum). When the convex PDMS membrane retracted back to a flat surface, it applied compressive gradient forces on the cell-laden hydrogels. The concentric circular hydrogel patterns confined the direction of hydrogel elongation, and the compressive strain on the hydrogel therefore resulted in elongation stretch in the radial direction to guide cell alignment. NIH3T3 cells were cultured in the chip for 3 days with compressive strains that varied from ~65% (center) to ~15% (boundary) on hydrogels. We found that the hydrogel geometry dominated the cell alignment near the outside boundary, where cells aligned along the circular direction, and the compressive strain dominated the cell alignment near the center, where cells aligned radially. This study developed a new and simple approach to facilitate cellular alignment based on hydrogel geometry and strain stimulation for tissue engineering applications. This platform offers unique advantages and is significantly different from the existing approaches owing to the fact that gradient generation was accomplished in a miniature device without using an external mechanical source.

25th anniversary article: Rational design and applications of hydrogels in regenerative medicine.

Hydrogels are hydrophilic polymer-based materials with high water content and physical characteristics that resemble the native extracellular matrix. Because of their remarkable properties, hydrogel systems are used for a wide range of biomedical applications, such as three-dimensional (3D) matrices for tissue engineering, drug-delivery vehicles, composite biomaterials, and as injectable fillers in minimally invasive surgeries. In addition, the rational design of hydrogels with controlled physical and biological properties can be used to modulate cellular functionality and tissue morphogenesis. Here, the development of advanced hydrogels with tunable physiochemical properties is highlighted, with particular emphasis on elastomeric, light-sensitive, composite, and shape-memory hydrogels. Emerging technologies developed over the past decade to control hydrogel architecture are also discussed and a number of potential applications and challenges in the utilization of hydrogels in regenerative medicine are reviewed. It is anticipated that the continued development of sophisticated hydrogels will result in clinical applications that will improve patient care and quality of life.

Electrospun PGS:PCL microfibers align human valvular interstitial cells and provide tunable scaffold anisotropy.

Tissue engineered heart valves (TEHV) can be useful in the repair of congenital or acquired valvular diseases due to their potential for growth and remodeling. The development of biomimetic scaffolds is a major challenge in heart valve tissue engineering. One of the most important structural characteristics of mature heart valve leaflets is their intrinsic anisotropy, which is derived from the microstructure of aligned collagen fibers in the extracellular matrix (ECM). In the present study, a directional electrospinning technique is used to fabricate fibrous poly(glycerol sebacate):poly(caprolactone) (PGS:PCL) scaffolds containing aligned fibers, which resemble native heart valve leaflet ECM networks. In addition, the anisotropic mechanical characteristics of fabricated scaffolds are tuned by changing the ratio of PGS:PCL to mimic the native heart valve's mechanical properties. Primary human valvular interstitial cells (VICs) attach and align along the anisotropic axes of all PGS:PCL scaffolds with various mechanical properties. The cells are also biochemically active in producing heart-valve-associated collagen, vimentin, and smooth muscle actin as determined by gene expression. The fibrous PGS:PCL scaffolds seeded with human VICs mimick the structure and mechanical properties of native valve leaflet tissues and would potentially be suitable for the replacement of heart valves in diverse patient populations.

Hydrogel surfaces to promote attachment and spreading of endothelial progenitor cells.

Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin-based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA-based hydrogels provides an attractive surface for EPCs promoting spreading and the formation of an endothelial monolayer on the hydrogel surface. To increase EPC adhesion and spreading, we covalently immobilized CD34 antibody (Ab) on HA-heparin hydrogels, using standard EDC/NHS amine-coupling strategies. We found that EPC adhesion and spreading on CD34 Ab-immobilized HA-heparin hydrogels was significantly higher than their non-modified analogues. Once adhered, EPCs spread and formed an endothelial layer on both non-modified and CD34 Ab-modified HA-heparin hydrogels after 3 days of culture. We did not observe significant adhesion and spreading when heparin was not included in the control hydrogels. In addition to EPCs, we also used human umbilical cord vein endothelial cells (HUVECs), which adhered and spread on HA-heparin hydrogels. Macrophages exhibited significantly less adhesion compared to EPCs on the same hydrogels. This composite material could possibly be used to develop surface coatings for artificial cardiovascular implants, due to its specificity for EPC and endothelial cells on an otherwise non-thrombogenic surface.