Tissue Engineering Design Research Articles

Atomic Force Microscopy: A Powerful Tool to Address Scaffold Design in Tissue Engineering.

Functional polymers currently represent a basic component of a large range of biological and biomedical applications including molecular release, tissue engineering, bio-sensing and medical imaging. Advancements in these fields are driven by the use of a wide set of biodegradable polymers with controlled physical and bio-interactive properties. In this context, microscopy techniques such as Atomic Force Microscopy (AFM) are emerging as fundamental tools to deeply investigate morphology and structural properties at micro and sub-micrometric scale, in order to evaluate the in time relationship between physicochemical properties of biomaterials and biological response. In particular, AFM is not only a mere tool for screening surface topography, but may offer a significant contribution to understand surface and interface properties, thus concurring to the optimization of biomaterials performance, processes, physical and chemical properties at the micro and nanoscale. This is possible by capitalizing the recent discoveries in nanotechnologies applied to soft matter such as atomic force spectroscopy to measure surface forces through force curves. By tip-sample local interactions, several information can be collected such as elasticity, viscoelasticity, surface charge densities and wettability. This paper overviews recent developments in AFM technology and imaging techniques by remarking differences in operational modes, the implementation of advanced tools and their current application in biomaterials science, in terms of characterization of polymeric devices in different forms (i.e., fibres, films or particles).

Design and Fabrication of Fibrous Nanomaterials Using Pull Spinning

The assembly of natural and synthetic polymers into fibrous nanomaterials has applications ranging from textiles, tissue engineering, photonics, and catalysis. However, rapid manufacturing of these materials is challenging, as the state of the art in nanofiber assembly remains limited by factors such as solution polarity, production rate, applied electric fields, or temperature. Here, the design and development of a rapid nanofiber manufacturing system termed pull spinning is described. Pull spinning is compact and portable, consisting of a high‐speed rotating bristle that dips into a polymer or protein reservoir and pulls a droplet from solution into a nanofiber. When multiple layers of nanofibers are collected, they form a nonwoven network whose composition, orientation, and function can be adapted to multiple applications. The capability of pull spinning to function as a rapid, point‐of‐use fiber manufacturing platform is demonstrated for both muscle tissue engineering and textile design.image

Effects of cell type and configuration on anabolic and catabolic activity in 3D co-culture of mesenchymal stem cells and nucleus pulposus cells.

ABSTRACTTissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co‐culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC‐only and co‐culture groups than in MSC‐only groups, with NPC‐only groups exhibiting the highest anabolic gene expression levels. However, NPC‐only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP‐1, MMP‐9, MMP‐13, and ADAMTS‐5). MSC‐only groups were unaffected by degenerative media conditions, and co‐culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co‐culture and NPC‐only groups. Co‐culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP‐13 and ADAMTS‐5 expression in hypoxic and inflammatory media conditions. Co‐culture micropellets were also found to self‐organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:61–73, 2017.

Fabrication and modelling of fractal, biomimetic, micro and nano-topographical surfaces

Natural surface topographies are often self-similar with hierarchical features at the micro and nanoscale, which may be mimicked to overcome modern tissue engineering and biomaterial design limitations. Specifically, a cell’s microenvironment within the human body contains highly optimised, fractal topographical cues, which directs precise cell behaviour. However, recreating biomimetic, fractal topographies in vitro is not a trivial process and a number of fabrication methods have been proposed but often fail to precisely control the spatial resolution of features at different lengths scales and hence, to provide true biomimetic properties. Here, we propose a method of accurately reproducing the self-similar, micro and nanoscale topography of a human biological tissue into a synthetic polymer through an innovative fabrication process. The biological tissue surface was characterised using atomic force microscopy (AFM) to obtain spatial data in X, Y and Z, which was converted into a grayscale ‘digital photomask’. As a result of maskless grayscale optical lithography followed by modified deep reactive ion etching and replica molding, we were able to accurately reproduce the fractal topography of acellular dermal matrix (ADM) into polydimethylsiloxane (PDMS). Characterisation using AFM at three different length scales revealed that the nano and micro-topographical features, in addition to the fractal dimension, of native ADM were reproduced in PDMS. In conclusion, it has been shown that the fractal topography of biological surfaces can be mimicked in synthetic materials using the novel fabrication process outlined, which may be applied to significantly enhance medical device biocompatibility and performance.

Sugary interfaces mitigate contact damage where stiff meets soft.

The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina's foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+–DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.

Architecture of the Corpus Spongiosum: An Anatomical Study

Urethral reconstruction is performed for urethral stricture or hypospadias correction. Research on urethral tissue engineering is increasing. Because the corpus spongiosum is important to support the urethra, urethral tissue engineering should ideally be combined with reconstruction of a corpus spongiosum. We describe a method to visualize and measure the architecture of the corpus spongiosum, which is needed for scaffold design. The penis was dissected from 2 unembalmed male cadavers. One penis was flaccid and the other was erect, as induced by saline infusion. Both were frozen in ice. At 6 sites sections were obtained in the transverse and frontal directions. After digitalizing the stained sections the images were edited, area measurements were taken and a 3-dimensional reconstruction was made. In transverse sections the mean area of the vascular lumen was 60% and 77% in the flaccid and the erect corpus spongiosum, and in frontal sections it was 53% and 74%, respectively. This indicated a 129% transverse increase and a 140% longitudinal increase in erection. Section sites did not essentially differ except in the glans penis. Frontal sections showed larger vascular cavities and more incomplete septae than transverse sections. This study provides what is to our knowledge novel information on corpus spongiosum architecture, which is relevant for scaffold design in tissue engineering. The study protocol can be used in future research with a larger number of specimens and more extensive analyses.

Matrix stiffness modulates formation and activity of neuronal networks of controlled architectures

The ability to construct easily in vitro networks of primary neurons organized with imposed topologies is required for neural tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. However, accumulating evidence suggests that the mechanical properties of the culture matrix can modulate important neuronal functions such as growth, extension, branching and activity. Here we designed robust and reproducible laminin-polylysine grid micropatterns on cell culture substrates that have similar biochemical properties but a 100-fold difference in Young's modulus to investigate the role of the matrix rigidity on the formation and activity of cortical neuronal networks. We found that cell bodies of primary cortical neurons gradually accumulate in circular islands, whereas axonal extensions spread on linear tracks to connect circular islands. Our findings indicate that migration of cortical neurons is enhanced on soft substrates, leading to a faster formation of neuronal networks. Furthermore, the pre-synaptic density was two times higher on stiff substrates and consistently the number of action potentials and miniature synaptic currents was enhanced on stiff substrates. Taken together, our results provide compelling evidence to indicate that matrix stiffness is a key parameter to modulate the growth dynamics, synaptic density and electrophysiological activity of cortical neuronal networks, thus providing useful information on scaffold design for neural tissue engineering.

Double thin film-based sandwich-cell carrier design for multicellular tissue engineering

Many organs are multicellular and each cell type requires a different microenvironment. Thus, there is a need for modular structures where the microenvironment of each cell type can be tuned separately. Herein, we describe enzymatically crosslinked gelatin based double layered film structures where each layer can be loaded with growth factors separately. As a model, we have developed a bi-layer system to produce a respiratory epithelium. This system constitutes an in vitro “epithelial patch” that can be adhered to the lumen of any implant. Crosslinking of the patches with transglutaminase resulted in 7days of stability at 37°C. The film layer was first used to release growth factors and it was shown that the release significantly improved the proliferation over 5days. A549 human lung epithelial cells were used and under the release of an epithelial growth supplement mix, there was a significant improvement on the epithelial proliferation (p<0.01). The designed substrate was successfully attached to titanium implants and we demonstrated the stability of the epithelial patch under in vitro conditions for 7days without deterioration. Under co-culture conditions for three days both cell types were alive. Such patches can be used to obtain fast epithelialization of large implant surfaces.

The microenvironment and cell behavior

Event Abstract Back to Event The microenvironment and cell behavior Vasif Hasirci1 1 Middle East Technical University, Biological Sciences, Türkiye Extracellular matrix and its organization are very critical for the function and viability of the tissues. Tissues are organized in a variety of complex forms and the current approach in design for biomaterials and tissue engineering takes this into account in order to understand the cell and material interactions and also to produce successful products. The nerves are organized in the form of fiber bundles and most of the current treatments of severed peripheral and central nervous system elements aim to provide a form of guiding element[1]. This could be a simple empty tube into which severed proximal and the distal nerve ends are introduced in order to support aligning and healing (Fig 1A). Another tissue consisting of highly aligned elements is the muscle[2]. An example of repair attempt to damaged muscle tissue after a heart attack is to employ a muscle patch with stem cells aligned on electrospun polymeric fibers prepared in the form of a mesh, a mat or a bundle (Fig 1B). When a systematic attempt is made to determine the factors that make a surface attractive or repulsive for cells, planned creation of surfaces highly decorated with topographical and chemical/biological features using micro- and nanotechnology methods. Topographical features such as micro and nano-sized posts/pillars and wells are designed for increasing attachment on the surface and also for forcing conformational changes in the cell and in their nuclei. Physical patterns might convince cells to adhere preferentially on certain regions or constrain them in voids or gaps much smaller than their normal sizes (Figs 1C , 1D)[3],[4]. There are also chemical and biological patterning approaches that are used to create adhesive and repulsive regions or islands and posts with tips modified with cell adhesive molecules such as fibronectin or cell repulsive molecules such as PEG. The results of these intense activities find application in other fields such as biosensors. Thus the design of the microenvironment of the cells is becoming a very important area in the field of biomaterials and tissue engineering and the newly evolving techniques contribute significantly to the advances made in these fields. Figure 1. Various extracellular surfaces designed and used in guidance of various tissues. A) Nerve guide[1], B) Cardiac patch[2], C) Selective attachment of cells on micropillared surfaces[3], D) Micropatterned surface with cells arranging their cytoskeleton to fit the pillar dimensions for bone tissue studies[4]. BIOMATEN for the Ministry of Development funds; METU Grants

Material and scaffold design and development for intervertebral disc tissue engineering and regeneration

Material and scaffold design and development for intervertebral disc tissue engineering and regeneration

Laminin matrix promotes hepatogenic terminal differentiation of human bone marrow mesenchymal stem cells.

The application of stem cells holds great promises in cell transplants. Considering the lack of optimal in vitro model for hepatogenic differentiation, this study was designed to examine the effects of laminin matrix on the improvement of in vitro differentiation of human bone marrow mesenchymal stem cells (hBM-MSC) into the more functional hepatocyte-like cells. Characterization of the hBM-MSCs was performed by immunophenotyping and their differentiation into the mesenchymal-derived lineage. Then, cells were seeded on the laminin-coated or tissue culture polystyrene (TCPS). The differentiation was carried out during two steps. Afterward, the expression of hepatocyte markers such as AFP, ALB, CK-18, and CK-19 as well as the expression of C-MET, the secretion of urea, and the activity of CYP3A4 enzyme were determined. Moreover, the cytoplasmic glycogen storage was examined by periodic acid-Schiff (PAS) staining. The results demonstrated that the culture of hBM-MSC on laminin considerably improved hepatogenic differentiation compared to TCP group. A significant elevated level of urea biosynthesis and CYP3A4 enzyme activity was observed in the media of the laminin-coated differentiated cells (P<0.05). Furthermore higher expressions of both AFP and ALB were determined in cells differentiated on laminin matrix. Glycogen accumulation was not detected in the undifferentiated hBM-MSCs, however, both differentiated cells in laminin and TCPS groups demonstrated the intracellular glycogen accumulation on day 21 of hepatogenic differentiation. Taken together, these findings may indicate that laminin matrix can improve terminal differentiation of hepatocyte-like cells from hBM-MSCs. Thus, laminin might be considered as a suitable coating in hepatic tissue engineering designs.

Interaction of Hyaluronan with Cationic Nanoparticles.

The polysaccharide hyaluronan (HA) is a main component of peri- and extracellular matrix, and an attractive molecule for materials design in tissue engineering and nanomedicine. Here, we study the morphology of complexes that form upon interaction of nanometer-sized amine-coated gold particles with this anionic, linear, and regular biopolymer in solution and grafted to a surface. We find that cationic nanoparticles (NPs) have profound effects on HA morphology on the molecular and supramolecular scale. Quartz crystal microbalance (QCM-D) shows that depending on their relative abundance, cationic NPs promote either strong compaction or swelling of films of surface-grafted HA polymers (HA brushes). Transmission electron and atomic force microscopy reveal that the NPs do also give rise to complexes of distinct morphologies-compact nanoscopic spheres and extended microscopic fibers-upon interaction with HA polymers in solution. In particular, stable and hydrated spherical complexes of single HA polymers with NPs can be prepared when balancing the ionizable groups on HA and NPs. The observed self-assembly phenomena could be useful for the design of drug delivery vehicles and a better understanding of the reorganization of HA-rich synthetic or biological matrices.

Multidimensional characterisation of biomechanical structures by combining Atomic Force Microscopy and Focused Ion Beam: A study of the rat whisker

Understanding the heterogeneity of biological structures, particularly at the micro/nano scale can offer insights valuable for multidisciplinary research in tissue engineering and biomimicry designs. Here we propose to combine nanocharacterisation tools, particularly Focused Ion Beam (FIB) and Atomic Force Microscopy (AFM) for three dimensional mapping of mechanical modulus and chemical signatures. The prototype platform is applied to image and investigate the fundamental mechanics of the rat face whiskers, a high-acuity sensor used to gain detailed information about the world. Grazing angle FIB milling was first applied to expose the interior cross section of the rat whisker sample, followed by a “lift-out” method to retrieve and position the target sample for further analyses. AFM force spectroscopy measurements revealed a non-uniform pattern of elastic modulus across the cross section, with a range from 0.8GPa to 13.5GPa. The highest elastic modulus was found at the outer cuticle region of the whisker, and values gradually decreased towards the interior cortex and medulla regions. Elemental mapping with EDS confirmed that the interior of the rat whisker is dominated by C, O, N, S, Cl and K, with a significant change of elemental distribution close to the exterior cuticle region. Based on these data, a novel comprehensive three dimensional (3D) elastic modulus model was constructed, and stress distributions under realistic conditions were investigated with Finite Element Analysis (FEA). The simulations could well account for the passive whisker deflections, with calculated resonant frequency as well as force–deflection for the whiskers being in good agreement with reported experimental data. Limitations and further applications are discussed for the proposed FIB/AFM approach, which holds good promise as a unique platform to gain insights on various heterogeneous biomaterials and biomechanical systems.

Development and evaluation of elastomeric hollow fiber membranes as small diameter vascular graft substitutes.

Engineering of small diameter (<6mm) vascular grafts (SDVGs) for clinical use remains a significant challenge. Here, elastomeric polyester urethane (PEU)-based hollow fiber membranes (HFMs) are presented as an SDVG candidate to target the limitations of current technologies and improve tissue engineering designs. HFMs are fabricated by a simple phase inversion method. HFM dimensions are tailored through adjustments to fabrication parameters. The walls of HFMs are highly porous. The HFMs are very elastic, with moduli ranging from 1-4MPa, strengths from 1-5MPa, and max strains from 300-500%. Permeability of the HFMs varies from 0.5-3.5×10(-6)cm/s, while burst pressure varies from 25 to 35psi. The suture retention forces of HFMs are in the range of 0.8 to 1.2N. These properties match those of blood vessels. A slow degradation profile is observed for all HFMs, with 71 to 78% of the original mass remaining after 8weeks, providing a suitable profile for potential cellular incorporation and tissue replacement. Both human endothelial cells and human mesenchymal stem cells proliferate well in the presence of HFMs up to 7days. These results demonstrate a promising customizable PEU HFMs for small diameter vascular repair and tissue engineering applications.

Effect of silk fibroin on degradation and in vivo biocompatibility of poly (L-lactic-co-ε-caprolactone) electronspun nanofibrous scaffolds

Objective To investigate the effect of silk fibroin(SF) on degradation and biocompatibility of poly(L-lactic acidco-e-caprolactone)(P[LLA-CL]) in vivo.Methods The scaffolds of P(LLA-CL)(w / w = 1 ∶ 1) blended with 25% of SF(SF / P[LLA-CL]) and P(LLA-CL) were prepared by electrospinning.Both kinds of scaffolds were subcutaneously implanted in 45 6-monthold rats for up to 6 months to evaluate their degradation and biocompatibility characteristics.Results Pathological sections showed P(LLA-CL) scaffold become swollen and began to separate into different layers after 3 months,and then become broken after 6 months;while SF / P(LLA-CL) scaffold largely maintained its structure after 6 months.Immunohistochemical staining showed a large number of macrophages on the surface and in P(LLA-CL) scaffolds 1 month after implantation,and they could still be found 3 months after implantation,accompanied by foreign body giant cells; while no obvious macrophages or foreign body giant cells were found in SF / P(LLA-CL) scaffolds at different time points.Examination of inflammatory gene expression showed that TNF-α and IL-10 expression in P(LLA-CL) scaffolds was significantly higher than that in SF / P(LLA-CL) scaffolds 1 week after implantation(P 0.05),the same was also true for TNF-α,IL-1β and IL-10 expression 1 month after implantation(P 0.05),for TNF-α and IL-10 expression 2 months after implantation(P 0.05),for TGF-β expression 3 months after implantation(P 0.05),and for IL-1β and TGF-β expression 6months after implantation(P 0.05).Conclusion SF incorporation can delay degradation,reduce inflammation,and improve the biocompatibility of P(LLA-CL) scaffolds,which may provide reference for scaffold design in tissue engineering.

The Application of Stem Cell Based Tissue Engineering in Spinal Cord Injury Repair.

Spinal Cord Injury (SCI) results in the permanent functional impairment, leading to monoplegia, paraplegia or tetraplegia with tremendous social and economic burden. The intrinsic repair mechanism has been proven to be insufficient. The complex pathophysiology after injury imposes enormous challenging to functional recovery, given the most advanced medical intervention nowadays. Therefore, the development of effective therapeutic strategy for spinal cord injury management is in great need. Here we review a stem cell based tissue engineering approach under preclinical or clinical development for spinal cord injury, with a focus on promoting functional recovery after SCI, aiming to provide some beneficial suggestion on stem cell based tissue engineering design.

Current strategies in multiphasic scaffold design for osteochondral tissue engineering: A review.

The repair of osteochondral defects requires a tissue engineering approach that aims at mimicking the physiological properties and structure of two different tissues (cartilage and bone) using specifically designed scaffold-cell constructs. Biphasic and triphasic approaches utilize two or three different architectures, materials, or composites to produce a multilayered construct. This article gives an overview of some of the current strategies in multiphasic/gradient-based scaffold architectures and compositions for tissue engineering of osteochondral defects. In addition, the application of finite element analysis (FEA) in scaffold design and simulation of in vitro and in vivo cell growth outcomes has been briefly covered. FEA-based approaches can potentially be coupled with computer-assisted fabrication systems for controlled deposition and additive manufacturing of the simulated patterns. Finally, a summary of the existing challenges associated with the repair of osteochondral defects as well as some recommendations for future directions have been brought up in the concluding section of this article.

Tissue regeneration in the pores of poly(lactide-co-glycolide)-impregnated wall of expanded polytetrafluoroethylene (ePTFE) hybrid grafts

Understanding of relationship between pore properties and tissue regeneration of expanded polytetrafluoroethylene (ePTFE) is important in design of vascular tissue engineering. Tissue regeneration into a micron scale pore of the ePTFE wall was investigated by employing techniques of superficial surface modification of ePTFE, fabrication of the hybrid scaffold composed of biodegradable poly (lactide-co-glycolide) (PLGA) and ePTFE, and seeding of vascular cells on its lumen surface. The ePTFE was in advance transformed into a hybrid scaffold by sequential four steps of treatments such as chemical modification of ePTFE surfaces, impregnation of biodegradable PLGA polymer into its wall pores, and coatings of both PLGA polymer on the ePTFE lumen surface and collagens on the PLGA-coated lumen surface. The hybrid scaffold was in advance in vitro tissue-cultured with vascular smooth muscle for 12 weeks and stem cells for another 2 weeks on its collagen-coated lumen surface, thus obtaining an in vitro tissue-cultured scaffold. This in vitro tissue-cultured hybrid scaffold was implanted in a carotid artery of mongrel dog for 4 weeks. The morphologies of the hybrid grafts explanted from the artery were analyzed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), focusing on tissue regeneration in the modified pores of ePTFE wall. They demonstrated migration of smooth muscle cells into the PLGA-impregnated/surface-modified pores of ePTFE wall along biodegradation of impregnated PLGA polymer, leading to tissue regeneration in its surface-modified pores. TEM results of the patent hybrid grafts showed both cell organelles and extracellular matrix of the regenerated media tissues in the pore channels of ePTFE wall with 20–30 μm inter-nodal distances.

Three-dimensionally printed biological machines powered by skeletal muscle.

Combining biological components, such as cells and tissues, with soft robotics can enable the fabrication of biological machines with the ability to sense, process signals, and produce force. An intuitive demonstration of a biological machine is one that can produce motion in response to controllable external signaling. Whereas cardiac cell-driven biological actuators have been demonstrated, the requirements of these machines to respond to stimuli and exhibit controlled movement merit the use of skeletal muscle, the primary generator of actuation in animals, as a contractile power source. Here, we report the development of 3D printed hydrogel "bio-bots" with an asymmetric physical design and powered by the actuation of an engineered mammalian skeletal muscle strip to result in net locomotion of the bio-bot. Geometric design and material properties of the hydrogel bio-bots were optimized using stereolithographic 3D printing, and the effect of collagen I and fibrin extracellular matrix proteins and insulin-like growth factor 1 on the force production of engineered skeletal muscle was characterized. Electrical stimulation triggered contraction of cells in the muscle strip and net locomotion of the bio-bot with a maximum velocity of ∼ 156 μm s(-1), which is over 1.5 body lengths per min. Modeling and simulation were used to understand both the effect of different design parameters on the bio-bot and the mechanism of motion. This demonstration advances the goal of realizing forward-engineered integrated cellular machines and systems, which can have a myriad array of applications in drug screening, programmable tissue engineering, drug delivery, and biomimetic machine design.

Enzyme Prodrug Therapy Engineered into Biomaterials

In this work, enzyme‐prodrug therapy (EPT) is engineered into hydrogel biomaterials to achieve localized synthesis of the drugs and their delivery to the adhering cells. The use of EPT in the context of drug delivery mediated by biomaterials significantly empowers the latter in that the same hydrogel is used to successfully synthesize several drugs with dissimilar structures and therapeutic effects. The concentration of the synthesized drugs is conveniently controlled by the concentration of the administered prodrugs. Using prodrugs for two therapeutic agents allows their synthesis and delivery with independent control over the concentration and the time of administration of each of the drugs. Using these tools, sequential delivery of drugs for anti‐inflammatory and anti‐proliferative activity is accomplished whereby the synthesis of drugs is mediated by the same enzyme‐functionalized hydrogel. The use of EPT to perform combination therapy mediated by an implantable biomaterial is also reported. Taken together, these results contribute significantly to the development of flexible and highly powerful tools of substrate‐mediated drug delivery with applications in the design of therapeutic implants and tissue engineering.