Biodegradable Poly(ester) Urethane Acrylate Resins for Digital Light Processing: From Polymer Synthesis to 3D Printed Tissue Engineering Constructs.

Digital light processing (DLP) is an accurate and fast additive manufacturing technique to produce a variety of products, from patient-customized biomedical implants to consumer goods. However, DLP's use in tissue engineering has been hampered due to a lack of biodegradable resin development. Herein, a library of biodegradable poly(esters) capped with urethane acrylate (with variations in molecular weight) is investigated as the basis for DLP printable resins for tissue engineering. The synthesized oligomers show good printability and are capable of creating complex structures with mechanical moduli close to those of medium-soft tissues (1-3 MPa). While fabricated films from different molecular weight resins show few differences in surface topology, wettability, and protein adsorption, the adhesion and metabolic activity of NCTC clone 929 (L929) cells and human dermal fibroblasts (HDFs) are significantly different. Resins from higher molecular weight oligomers provide greater cell adhesion and metabolic activity. Furthermore, these materials show compatibility in a subcutaneous in vivo pig model. These customizable, biodegradable, and biocompatible resins show the importance of molecular tuning and open up new possibilities for the creation of biocompatible constructs for tissue engineering.

Aggregation and Gelation Behavior of Stereocomplexed Four-Arm PLA-PEG Copolymers Containing Neutral or Cationic Linkers.

Poly(lactide) (PLA) and poly(ethylene glycol) (PEG)-based hydrogels were prepared by mixing phosphate buffer saline (PBS, pH 7.4) solutions of four-arm (PEG-PLA)2-R-(PLA-PEG)2 enantiomerically pure copolymers having the opposite chirality of the poly(lactide) blocks. Dynamic Light Scattering, rheology measurements, and fluorescence spectroscopy suggested that, depending on the nature of the linker R, the gelation process followed rather different mechanisms. In all cases, mixing of equimolar amounts of the enantiomeric copolymers led to micellar aggregates with a stereocomplexed PLA core and a hydrophilic PEG corona. Yet, when R was an aliphatic heptamethylene unit, temperature-dependent reversible gelation was mainly induced by entanglements of PEG chains at concentrations higher than 5 wt.%. When R was a linker containing cationic amine groups, thermo-irreversible hydrogels were promptly generated at concentrations higher than 20 wt.%. In the latter case, stereocomplexation of the PLA blocks randomly distributed in micellar aggregates is proposed as the major determinant of the gelation process.

Enzymatic co-crosslinking of star-shaped poly(ethylene glycol) tyramine and hyaluronic acid tyramine conjugates provides elastic biocompatible and biodegradable hydrogels.

A combination of the viscoelastic properties of hyaluronic acid (HA) and the elastic properties of star shaped 8-arm poly(ethylene glycol) (8-arm PEG) was used to design in-situ forming hydrogels. Hydrogels were prepared by the enzymatic crosslinking of a partially tyramine modified 8-arm PEG and a tyramine conjugated HA using horseradish peroxidase in the presence of hydrogen peroxide. Hydrogels of the homopolymer conjugates and mixtures thereof were rapidly formed within seconds under physiological conditions at low polymer and enzyme concentrations. Elastic hydrogels with high gel content (≥95%) and high storage moduli (up to 22.4 kPa) were obtained. An in vitro study in the presence of hyaluronidase (100 U/mL) revealed that with increasing PEG content the degradation time of the hybrid hydrogels increased up to several weeks, whereas hydrogels composed of only hyaluronic acid degraded within 2 weeks. Human mesenchymal stem cells (hMSCs) incorporated in the hybrid hydrogels remained viable as shown by a PrestoBlue and a live-dead assay, confirming the biocompatibility of the constructs. The production of an extracellular matrix by re-differentiation of encapsulated human chondrocytes was followed over a period of 28 days. Gene expression indicated that these highly elastic hydrogels induced an enhanced production of collagen type II. At low PEG-TA/HA-TA ratios a higher expression of SOX 9 and ACAN was observed. These results indicate that by modulating the ratio of PEG/HA, injectable hydrogels can be prepared applicable as scaffolds for tissue regeneration applications.

Installation of click-type functional groups enable the creation of an additive manufactured construct for the osteochondral interface.

Melt extrusion-based additive manufacturing (AM) is often used to fabricate scaffolds for osteochondral (OC) regeneration. However, there are two shortcomings associated with this scaffold manufacturing technique for engineering of tissue interfaces: (a) most polymers used in the processing are bioinert, and (b) AM scaffolds often contain discrete (material) gradients accompanied with mechanically weak interfaces. The inability to mimic the gradual transition from cartilage to bone in OC tissue leads to poor scaffold performance and even failure. We hypothesized that introducing peptide gradients on the surface could gradually guide human mesenchymal stromal cell (hMSC) differentiation, from a chondrogenic towards on osteogenic phenotype. To work towards this goal, we initially manufactured poly(ϵ-caprolactone)-azide (PCLA) and PCL-maleimide (PCLM) scaffolds. The surface exposed click-type functional groups, with a surface concentration in the 102pmol cm-2regime, were used to introduce bone morphogenic protein-2 or transforming growth factor-beta binding peptide sequences to drive hMSC differentiation towards osteogenic or chondrogenic phenotypes, respectively. After 3 weeks of culture in chondrogenic medium, we observed differentiation towards hypertrophic chondrogenic phenotypes with expression of characteristic markers such as collagen X. In osteogenic medium, we observed the upregulation of mineralization markers. In basic media, the chondro-peptide displayed a minor effect on chondrogenesis, whereas the osteo-peptide did not affect osteogenesis. In a subcutaneous rat model, we observed a minimal foreign body response to the constructs, indicating biocompatibility. As proof-of-concept, we finally used a novel AM technology to showcase its potential to create continuous polymer gradients (PCLA and PCLM) across scaffolds. These scaffolds did not display delamination and were mechanically stronger compared to discrete gradient scaffolds. Due to the versatility of the orthogonal chemistry applied, this approach provides a general strategy for the field; we could anchor other tissue specific cues on the clickable groups, making these gradient scaffolds interesting for multiple interfacial tissue applications.

Injectable Cell-Laden Polysaccharide Hydrogels: In Vivo Evaluation of Cartilage Regeneration.

Previously, 5% w/v hyaluronic acid-tyramine (HA-TA) and dextran-tyramine (Dex-TA) enzymatically cross-linked hybrid hydrogels were demonstrated to provide a mechanically stable environment, maintain cell viability, and promote cartilaginous-specific matrix deposition in vitro. In this study, 5% w/v hybrid hydrogels were combined with human mesenchymal stem cells (hMSCs), bovine chondrocytes (bCHs), or a combination of both in a 4:1 ratio and subcutaneously implanted in the backs of male and female nude rats to assess the performance of cell-laden hydrogels in tissue formation. Subcutaneous implantation of these biomaterials showed signs of integration of the gels within the host tissue. Histological analysis showed residual fibrotic capsules four weeks after implantation. However, enhanced tissue invasion and some giant cell infiltration were observed in the HA-TA/Dex-TA hydrogels laden with either hMSCs or bCHs but not with the co-culture. Moreover, hMSC-bCH co-cultures showed beneficial interaction with the hydrogels, for instance, in enhanced cell proliferation and matrix deposition. In addition, we provide evidence that host gender has an impact on the performance of bCHs encapsulated in HA-TA/Dex-TA hydrogels. This study revealed that hydrogels laden with different types of cells result in distinct host responses. It can be concluded that 5% w/v hydrogels with a higher concentration of Dex-TA (≥50%) laden with bCH-hMSC co-cultures are adequate for injectable applications and in situ cell delivery in cartilage regeneration approaches.

Aerobic training increases mitochondrial respiratory capacity in human skeletal muscle stem cells from sedentary individuals.

The impact of aerobic training on human skeletal muscle cell (HSkMC) mitochondrial metabolism is a significant research gap, critical to understanding the mechanisms by which exercise augments skeletal muscle metabolism. We therefore assessed mitochondrial content and capacity in fully differentiated CD56+ HSkMCs from lean active (LA) and sedentary individuals with obesity (OS) at baseline, as well as lean/overweight sedentary individuals (LOS) at baseline and following an 18-day aerobic training intervention. Participants had in vivo skeletal muscle PCr recovery rate by 31P-MRS (mitochondrial oxidative kinetics) and cardiorespiratory fitness (V̇o2max) assessed at baseline. Biopsies of the vastus lateralis were performed for the isolation of skeletal muscle stem cells. LOS individuals repeated all assessments posttraining. HSkMCs were evaluated for mitochondrial respiratory capacity by high-resolution respirometry. Data were normalized to two indices of mitochondrial content (CS activity and OXPHOS protein expression) and a marker of total cell count (quantity of DNA). LA individuals had significantly higher V̇o2max than OS and LOS-Pre training; however, no differences were observed in skeletal muscle mitochondrial capacity, nor in carbohydrate- or fatty acid-supported HSkMC respiratory capacity. Aerobic training robustly increased in vivo skeletal muscle mitochondrial capacity of LOS individuals, as well as carbohydrate-supported HSkMC respiratory capacity. Indices of mitochondrial content and total cell count were similar among the groups and did not change with aerobic training. Our findings demonstrate that bioenergetic changes induced with aerobic training in skeletal muscle in vivo are retained in HSkMCs in vitro without impacting mitochondrial content, suggesting that training improves intrinsic skeletal muscle mitochondrial capacity.

Tethering Cells via Enzymatic Oxidative Crosslinking Enables Mechanotransduction in Non-Cell-Adhesive Materials.

Cell-matrix interactions govern cell behavior and tissue function by facilitating transduction of biomechanical cues. Engineered tissues often incorporate these interactions by employing cell-adhesive materials. However, using constitutively active cell-adhesive materials impedes control over cell fate and elicits inflammatory responses upon implantation. Here, an alternative cell-material interaction strategy that provides mechanotransducive properties via discrete inducible on-cell crosslinking (DOCKING) of materials, including those that are inherently non-cell-adhesive, is introduced. Specifically, tyramine-functionalized materials are tethered to tyrosines that are naturally present in extracellular protein domains via enzyme-mediated oxidative crosslinking. Temporal control over the stiffness of on-cell tethered 3D microniches reveals that DOCKING uniquely enables lineage programming of stem cells by targeting adhesome-related mechanotransduction pathways acting independently of cell volume changes and spreading. In short, DOCKING represents a bioinspired and cytocompatible cell-tethering strategy that offers new routes to study and engineer cell-material interactions, thereby advancing applications ranging from drug delivery, to cell-based therapy, and cultured meat.

Engineering of Optimized Hydrogel Formulations for Cartilage Repair.

The ideal scaffold for cartilage regeneration is expected to provide adequate mechanical strength, controlled degradability, adhesion, and integration with the surrounding native tissue. As it does this, it mimics natural ECMs functions, which allow for nutrient diffusion and promote cell survival and differentiation. Injectable hydrogels based on tyramine (TA)-functionalized hyaluronic acid (HA) and dextran (Dex) are a promising approach for cartilage regeneration. The properties of the hydrogels used in this study were adjusted by varying polymer concentrations and ratios. To investigate the changes in properties and their effects on cellular behavior and cartilage matrix formation, different ratios of HA- and dextran-based hybrid hydrogels at both 5 and 10% w/v were prepared using a designed mold to control generation. The results indicated that the incorporation of chondrocytes in the hydrogels decreased their mechanical properties. However, rheological and compression analysis indicated that 5% w/v hydrogels laden with cells exhibit a significant increase in mechanical properties after 21 days when the constructs are cultured in a chondrogenic differentiation medium. Moreover, compared to the 10% w/v hydrogels, the 5% w/v hybrid hydrogels increased the deposition of the cartilage matrix, especially in constructs with a higher Dex-TA content. These results indicated that 5% w/v hybrid hydrogels with 25% HA-TA and 75% Dex-TA have a high potential as injectable scaffolds for cartilage tissue regeneration.

In vitro degradation profiles and in vivo biomaterial-tissue interactions of microwell array delivery devices.

To effectively apply microwell array cell delivery devices their biodegradation rate must be tailored towards their intended use and implantation location. Two microwell array devices with distinct degradation profiles, either suitable for the fabrication of retrievable systems in the case of slow degradation, or cell delivery systems capable of extensive remodeling using a fast degrading polymer, were compared in this study. Thin films of a poly(ethylene glycol)-poly(butylene terephthalate) (PEOT-PBT) and a poly(ester urethane) were evaluated for their in vitro degradation profiles over 34 weeks incubation in PBS at different pH values. The PEOT-PBT films showed minimal in vitro degradation over time, while the poly(ester urethane) films showed extensive degradation and fragmentation over time. Subsequently, microwell array cell delivery devices were fabricated from these polymers and intraperitoneally implanted in Albino Oxford rats to study their biocompatibility over a 12-week period. The PEOT-PBT implants shown to be capable to maintain the microwell structure over time. Implants provoked a foreign body response resulting in multilayer fibrosis that integrated into the surrounding tissue. The poly(ester urethane) implants showed a loss of the microwell structures over time, as well as a fibrotic response until the onset of fragmentation, at least 4 weeks post implantation. It was concluded that the PEOT-PBT implants could be used as retrievable cell delivery devices while the poly(ester urethane) implants could be used for cell delivery devices that require remodeling within a 4-12 week period.

Multivalency Enables Dynamic Supramolecular Host-Guest Hydrogel Formation.

Supramolecular and dynamic biomaterials hold promise to recapitulate the time-dependent properties and stimuli-responsiveness of the native extracellular matrix (ECM). Host-guest chemistry is one of the most widely studied supramolecular bonds, yet the binding characteristics of host-guest complexes (ß-CD/adamantane) in relevant biomaterials have mostly focused on singular host-guest interactions or nondiscrete multivalent pendent polymers. The stepwise synergistic effect of multivalent host-guest interactions for the formation of dynamic biomaterials remains relatively unreported. In this work, we study how a series of multivalent adamantane (guest) cross-linkers affect the overall binding affinity and ability to form supramolecular networks with alginate-CD (Alg-CD). These binding constants of the multivalent cross-linkers were determined via NMR titrations and showed increases in binding constants occurring with multivalent constructs. The higher multivalent cross-linkers enabled hydrogel formation; furthermore, an increase in binding and gelation was observed with the inclusion of a phenyl spacer to the cross-linker. A preliminary screen shows that only cross-linking Alg-CD with an 8-arm-multivalent guest results in robust gel formation. These cytocompatible hydrogels highlight the importance of multivalent design for dynamically cross-linked hydrogels. These materials hold promise for development toward cell- and small molecule-delivery platforms and allow discrete and fine-tuning of network properties.

Developing hyaluronic acid microgels for sustained delivery of platelet lysate for tissue engineering applications.

Platelet lysate (PL), a blood product that contains high concentrations of growth factors (GFs), can be considered as a cost-effective source of multiple GFs. In this study, hyaluronic acid (HA) based microgels were developed for delivery of PL proteins. Spherical microgel were prepared using a water in oil emulsion method. First, hyaluronic acid was grafted with tyramine groups, after which prepared microdroplets were crosslinked via an enzymatic reaction in the presence of hydrogen peroxide and horseradish peroxidase. Because of electrostatic interactions, these microgels are promising carriers for positively charged proteins entrapment like most of the GFs. When microgels are incubated in PL solution, protein loading takes place which is mainly governed by nonspecific adsorption of plasma proteins. Although this hampered loading efficiency, loading could be increased by repeated washing and incubation steps. The loaded microgels presented a sustained release of PL growth factors for a period of two weeks. When PL enriched microgels were embedded in a HA bulk hydrogel, cell proliferation was higher compared to constructs without microgels. These findings suggest that the developed microgels are a potential candidate for sustained delivery of PL growth factors and present a solution to the issue of their short half-lives in vivo.

Spatiotemporal material functionalization via competitive supramolecular complexation of avidin and biotin analogs.

Spatiotemporal control over engineered tissues is highly desirable for various biomedical applications as it emulates the dynamic behavior of natural tissues. Current spatiotemporal biomaterial functionalization approaches are based on cytotoxic, technically challenging, or non-scalable chemistries, which has hampered their widespread usage. Here we report a strategy to spatiotemporally functionalize (bio)materials based on competitive supramolecular complexation of avidin and biotin analogs. Specifically, an injectable hydrogel is orthogonally post-functionalized with desthiobiotinylated moieties using multivalent neutravidin. In situ exchange of desthiobiotin by biotin enables spatiotemporal material functionalization as demonstrated by the formation of long-range, conformal, and contra-directional biochemical gradients within complex-shaped 3D hydrogels. Temporal control over engineered tissue biochemistry is further demonstrated by timed presentation and sequestration of growth factors using desthiobiotinylated antibodies. The method's universality is confirmed by modifying hydrogels with biotinylated fluorophores, peptides, nanoparticles, enzymes, and antibodies. Overall, this work provides a facile, cytocompatible, and universal strategy to spatiotemporally functionalize materials.

An injectable platelet lysate-hyaluronic acid hydrogel supports cellular activities and induces chondrogenesis of encapsulated mesenchymal stem cells.

Developing scaffolds that can provide cells and biological cues simultaneously in the defect site is of interest in tissue engineering field. In this study, platelet lysate (PL) as an autologous and inexpensive source of growth factors was incorporated into a cell-laden injectable hyaluronic acid-tyramine (HA-TA) hydrogel. Subsequently, the effect of platelet lysate on cell attachment, viability and differentiation of human mesenchymal stem cell (hMSCs) toward chondrocytes was investigated. HA-TA conjugates having a degree of substitution of 20 TA moieties per 100 disaccharide units were prepared and crosslinked in the presence of horseradish peroxidase and low concentrations of hydrogen peroxide. The storage moduli of the gels ranged from 500 to 2000 Pa and increased with increasing polymer concentration. In contrast to a retained round shape of the cells when using pure HA-TA hydrogel, the hMSCs attached and spread out in PL enriched matrix. The enrichment of hMSCs laden HA-TA hydrogels with PL induced a cartilage like extra cellular matrix deposition in vitro. The hMSCs increasingly deposited collagen type II and proteoglycans over time. The deposition of the new extracellular matrix (ECM) is simultaneous with gel degradation and resulted ultimately in the formation of a tough dense matrix. These findings demonstrate the potential of injectable HA-TA-PL hydrogel as a cell delivery system for cartilage regeneration. STATEMENT OF SIGNIFICANCE: Cartilage tissue has limited ability to self-repair because of its avascular nature. To have an efficient cartilage tissue regeneration, we combined platelet lysate (PL), as an autologous and inexpensive source of growth factors, with an injectable hyaluronic acid tyramine (HA-TA) hydrogel scaffold. Platelet lysate had a vital role in supporting human mesenchymal stem cells (hMSCs) activities, like cell attachment, viability and proliferation in the 3D hydrogel structure. Also, the hMSCs encapsulated HA-TA induced hyaline cartilage generation when placed in chondrogenic differentiation medium. This study introduces a new system for cartilage tissue engineering, which can be injected in a minimally invasive manner and is rich with patient's own growth factors and biological cues.

Kinetically stable metal ligand charge transfer complexes as crosslinks in nanogels/hydrogels: Physical properties and cytotoxicity.

A terpyridine end-functionalized 8-arm poly(ethylene glycol) was prepared using the reaction of a 4'-aminopentanoxy substituted terpyridine with a p-nitrophenyl chloroformate activated PEG-(OH)8. Supramolecular complexation of the polymer terpyridine moieties by Fe(2+) ions was investigated using NMR, UV-Vis and dynamic light scattering experiments. At low concentrations addition of Fe(2+) ions to an aqueous solution of the polymer conjugate afforded nanogels with a single size distribution around 250 nm. At concentrations above 3 wt%, and at a 1:2 metal to ligand molar ratio, hydrogels were formed with increasing mechanical properties at increasing polymer concentrations. Using bovine chondrocytes, the biocompatibility and potential cytotoxicity of the polymer conjugate, nanogels and hydrogels were studied. The polymer conjugate with free ligands was toxic to the cells likely due to depletion of essential metal ions. When the terpyridine groups were complexed with Fe(2+) ions, both nanogel suspensions and hydrogels showed no cytotoxicity in direct contact with chondrocytes. Indirect contact of gels with chondrocytes using transwells revealed the absence of toxic components by leaching. A Live-Dead assay on chondrocytes encapsulated in the hydrogels indicated that the hydrogels are cytocompatible, revealing the potential use of these materials for biomedical and pharmaceutical applications. STATEMENT OF SIGNIFICANCE: The binding between transition metal ions and ligands with multiple binding sites can be almost as strong as covalent bonds. This metal-ligand charge transfer (MLCT) complexation was used to crosslink water soluble polymers into hydrogels. This approach to novel materials may find applications in the biomedical and pharmaceutical fields. Transition metal ions are essential trace elements present in tissue but up to now no cytotoxicity data of free ligands are available. Data presented show that free ligands are toxic to cells likely by depletion of trace metal ions, whereas kinetically stable complexes are not cytotoxic even when embedded in hydrogels. These results provide fundamental issues to be considered in the design of hydrogels crosslinked through metal ligand complexation.

Hydrogels by supramolecular crosslinking of terpyridine end group functionalized 8-arm poly(ethylene glycol).

Metallo supramolecular assemblies of an 8-arm poly(ethylene glycol) partially substituted with terpyridyl end-groups and the transition metal ions Ni(2+), Fe(2+), Co(2+) and Zn(2+) were studied for their nano-particle formation at dilute conditions and gelation at higher concentrations. The large differences in dissociation rate constants of the metal ligand complexes largely determine the assembly behavior. Thermodynamically stable complexes are generated with Ni(2+) and Fe(2+) chlorides, which lead to distinct particle sizes of ∼200 nm in dilute conditions. The Co(2+) and Zn(2+) chlorides provide multiple size distributions revealing that mono and bis-complexes are present at equilibrium. Upon complexation, terpyridyl groups move to the outer sphere giving aggregates with a charged surface. At polymer concentrations above 5 wt%, crosslinking upon addition of transition metal ions provides hydrogels. Elastic hydrogels were obtained with Ni(2+), Fe(2+) and Co(2+) having storage moduli in excess of 20 kPa, whereas Zn(2+) gels are relatively viscous. Only Zn(2+) gels show a thermoreversible sol to gel transition at a temperature of 25 °C independent of polymer concentration.

Hydrogels in a historical perspective: from simple networks to smart materials.

Over the past decades, significant progress has been made in the field of hydrogels as functional biomaterials. Biomedical application of hydrogels was initially hindered by the toxicity of crosslinking agents and limitations of hydrogel formation under physiological conditions. Emerging knowledge in polymer chemistry and increased understanding of biological processes resulted in the design of versatile materials and minimally invasive therapies. Hydrogel matrices comprise a wide range of natural and synthetic polymers held together by a variety of physical or chemical crosslinks. With their capacity to embed pharmaceutical agents in their hydrophilic crosslinked network, hydrogels form promising materials for controlled drug release and tissue engineering. Despite all their beneficial properties, there are still several challenges to overcome for clinical translation. In this review, we provide a historical overview of the developments in hydrogel research from simple networks to smart materials.

Fluorescently labeled branched polymers and thermal responsive nanoparticles for live cell imaging.

Branched poly(methoxy-PEG acrylate) and thermally responsive poly(methoxy-PEG acrylate)-block-poly(N-isopropylacrylamide) are synthesized by RAFT polymerization. After reduction, these polymers are fluorescently labeled by reacting the free thiol groups with N-(5-fluoresceinyl)maleimide. As shown by DLS, the labeled copolymer poly(methoxy-PEG acrylate)-block-poly(N-isopropylacrylamide) forms nanoparticles at body temperature (37 °C) due to the presence of the thermosensitive poly(N-isopropylacrylamide). These materials were used as bioprobes for imaging HUVECs in vitro and chick embryo CAM in vivo. Both labeled polymer and nanoparticles are biocompatible and can be used as efficient fluorescent bioprobes.

The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells.

Self-attaching and cell-attracting in-situ forming dextran-tyramine conjugates hydrogels for arthroscopic cartilage repair.

Small cartilage defects are frequently treated with debridement or left untreated, predisposing to early onset osteoarthritis. We propose to fill these defects with a cell-free injectable hydrogel comprising dextran-tyramine conjugates (Dex-TA) that can be applied during arthroscopic procedures. In this study, we report on the adhesion mechanism between cartilage and Dex-TA hydrogels and enhancement of cell ingrowth by incorporation of Heparin-tyramine (Hep-TA) conjugates. The enzyme-catalyzed crosslinking reaction of Dex-TA and Hep-TA hydrogels is based on covalent bonding of hydroxyphenyl residues. We hypothesized that this reaction results in covalent bonding of the hydroxyphenyl residues in Dex-TA and Hep-TA to tyrosine residues in cartilage matrix proteins. The involvement of TA residues was confirmed by modelling the enzymatic reaction occurring during gelation. The mechanical analysis indicated that higher tyramine content led to stronger binding. Interfacial cartilage-hydrogel morphology and Raman spectroscopy demonstrated collagens' reorganization and evidenced the coupling of TA to tyrosine residues in collagen. Moreover, the addition of Hep-TA induced cell recruitment. Collectively, in vitro and ex vivo functional studies evidenced the covalent bonding of TA-containing hydrogels to tyrosine residues in cartilaginous matrix proteins. Moreover, the cell-attracting ability of these hydrogels could be explored to guide tissue repair in focal cartilage defects, preventing or delaying the onset osteoarthritis.

Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue engineering.

State-of-the-art bioactive hydrogels can easily and efficiently be formed by enzyme-catalyzed mild-crosslinking reactions in situ. Yet this cell-friendly and substrate-specific method remains under explored. Hydrogels prepared by using enzyme systems like tyrosinases, transferases and lysyl oxidases show interesting characteristics as dynamic scaffolds and as systems for controlled release. Increased attention is currently paid to hydrogels obtained via crosslinking of precursors by transferases or peroxidases as catalysts. Enzyme-mediated crosslinking has proven its efficiency and attention has now shifted to the development of enzymatically crosslinked hydrogels with higher degrees of complexity, mimicking extracellular matrices. Moreover, bottom-up approaches combining biocatalysts and self-assembly are being explored for the development of complex nano-scale architectures. In this review, the use of enzymatic crosslinking for the preparation of hydrogels as an innovative alternative to other crosslinking methods, such as the commonly used UV-mediated photo-crosslinking or physical crosslinking, will be discussed. Photo-initiator-based crosslinking may induce cytotoxicity in the formed gels, whereas physical crosslinking may lead to gels which do not have sufficient mechanical strength and stability. These limitations can be overcome using enzymes to form covalently crosslinked hydrogels. Herewith, we report the mechanisms involved and current applications, focusing on emerging strategies for tissue engineering and regenerative medicine.