Well-Defined Synthetic Copolymers with Pendant Aldehydes Form Biocompatible Strain-Stiffening Hydrogels and Enable Competitive Ligand Displacement.

Dynamic hydrogels are attractive platforms for tissue engineering and regenerative medicine due to their ability to mimic key extracellular matrix (ECM) mechanical properties like strain-stiffening and stress relaxation while enabling enhanced processing characteristics like injectability, 3D printing, and self-healing. Systems based on imine-type dynamic covalent chemistry (DCvC) have become increasingly popular. However, most reported polymers comprising aldehyde groups are based on either end-group-modified synthetic or side-chain-modified natural polymers; synthetic versions of side-chain-modified polymers are noticeably absent. To facilitate access to new classes of dynamic hydrogels, we report the straightforward synthesis of a water-soluble copolymer with a tunable fraction of pendant aldehyde groups (12-64%) using controlled radical polymerization and their formation into hydrogel biomaterials with dynamic cross-links. We found the polymer synthesis to be well-controlled with the determined reactivity ratios consistent with a blocky gradient microarchitecture. Subsequently, we observed fast gelation kinetics with imine-type cross-linking. We were able to vary hydrogel stiffness from ≈2 to 20 kPa, tune the onset of strain-stiffening toward a biologically relevant regime (σc ≈ 10 Pa), and demonstrate cytocompatibility using human dermal fibroblasts. Moreover, to begin to mimic the dynamic biochemical nature of the native ECM, we highlight the potential for temporal modulation of ligands in our system to demonstrate ligand displacement along the copolymer backbone via competitive binding. The combination of highly tunable composition, stiffness, and strain-stiffening, in conjunction with spatiotemporal control of functionality, positions these cytocompatible copolymers as a powerful platform for the rational design of next-generation synthetic biomaterials.

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.