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.

The ability to modulate stem cell differentiation in a three dimensional (3D) microenvironment for bone tissue engineering in absence of exogenous pharmaceutical agents such as bone morphogenic protein (BMP-2) remains a challenge. In this study, we introduce extracellular matrix (ECM)-mimicking nanocomposite hydrogels to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) for bone regeneration in absence of any osteoinducting factors. In particular, we have reinforced photocrosslinkable collagen-based matrix (gelatin methacryloyl, GelMA) used disk-shaped nanosilicates (nSi), a new class of two-dimensional (2D) nanomaterials. We show that nanoengineered hydrogels supported migration and proliferation of encapsulated hMSCs, with no signs of cell apoptosis or inflammatory cytokine responses. The addition of nSi significantly enhances osteogenic differentiation of encapsulated hMSCs as evident by the increase in alkaline phosphates (ALP) activity and deposition of biomineralized matrix compared to GelMA without nSi. We also show that microfabricated nanoengineered microgels can be used to pattern and control cellular behaviour. Furthermore, we also show that nanoengineered hydrogel have high biocompatibility as determined by in vivo experiments using immunocompetent rat model. Specifically, the hydrogels showed minimum localized immune responses, indicating it ability for tissue engineering applications. Overall, we showed the ability of nanoengineered hydrogels loaded with 2D nanosilicates for osteogenic differentiation of stem cells in vitro, in absence of any growth factors such as BMP-2. Our in vivo studies show high biocompatibility of nanocomposites and show the potential for growth factor free bone regeneration.

Isoelectric focusing (IEF) of type-I collagen molecules is a technology with proven efficacy to produce dense and aligned collagen-based biomaterials. The forces and mechanisms during IEF of collagen molecules in carrier ampholyte-free environments remain unknown. This study presents theoretical framework describing the congregation of collagen molecules along the isoelectric point (pI). A single molecule was modeled as a rod-like particle, distributed homogeneously between parallel electrodes. Upon application of electrical current, molecules migrated to the pI. The results showed that self-aggregation of collagen molecules along the pI occurred due to formation of a non-linear pH gradient that rendered the anodic side acidic, and the cathodic side basic. This pH profile and the amphoteric nature of collagen resulted in positively charged molecules at the anode and negatively charged molecules at the cathode. Therefore, repulsive electrostatic forces aided self-aggregation of molecules along the pI. The model could effectively validate the pI of collagen, the pI location, and predict that the instantaneous velocity acting on a molecule at the anode was higher than those velocities at the cathode. This fundamental information represents the baseline theory upon which we can expand our knowledge to the production of biomaterials to engineer soft tissues.

Collagen-based biomaterials are a viable option for tendon reconstruction and repair. However, the weak mechanical strength of collagen constructs is a major limitation. We have previously reported a novel methodology to form highly oriented electrochemically aligned collagen (ELAC) threads with mechanical properties converging on those of the natural tendon. In this study, we assessed the in vivo response of rabbit patellar tendon (PT) to braided ELAC bioscaffolds. Rabbit PTs were incised longitudinally and the ELAC bioscaffold was inlaid in one limb along the length of the tendon. The contralateral limb served as the sham-operated control. Rabbits were euthanized at 4 or 8 months postoperatively. High-resolution radiographs revealed the absence of ectopic bone formation around the bioscaffolds. Four months post-implantation, the histological sections showed that the ELAC bioscaffold underwent limited degradation and was associated with a low-grade granulomatous inflammation. Additionally, quantitative histology revealed that the cross-sectional areas of PTs with the ELAC bioscaffold were 29% larger compared with the controls. Furthermore, ELAC-treated PTs were significantly stiffer compared with the controls. The volume fraction of the tendon fascicle increased in the ELAC-treated PT compared with the controls. By 8 months, the ELAC bioscaffold was mostly absorbed and the enlargement in the area of tendons with implants subsided along with the resolution of the granulomatous inflammation. We conclude that ELAC is biocompatible and biodegradable and has the potential to be used as a biomaterial for tendon tissue engineering applications.

The musculoskeletal system is essential for maintaining posture, protecting organs, facilitating locomotion, and regulating various cellular and metabolic functions. Injury to this system due to trauma or wear is common, and severe damage may require surgery to restore function and prevent further harm. Autografts are the current gold standard for the replacement of lost or damaged tissues. However, these grafts are constrained by limited supply and donor site morbidity. Allografts, xenografts, and alloplastic materials represent viable alternatives, but each of these methods also has its own problems and limitations. Technological advances in three-dimensional (3D) printing and its biomedical adaptation, 3D bioprinting, have the potential to provide viable, autologous tissue-like constructs that can be used to repair musculoskeletal defects. Though bioprinting is currently unable to develop mature, implantable tissues, it can pattern cells in 3D constructs with features facilitating maturation and vascularization. Further advances in the field may enable the manufacture of constructs that can mimic native tissues in complexity, spatial heterogeneity, and ultimately, clinical utility. This review studies the use of 3D bioprinting for engineering bone, cartilage, muscle, tendon, ligament, and their interface tissues. Additionally, the current limitations and challenges in the field are discussed and the prospects for future progress are highlighted.

A key step during the synthesis of collagen constructs is the incubation of monomeric collagen in phosphate buffer saline (PBS) to promote fibrillogenesis in the collagen network. Optimal PBS treatment conditions for monomeric collagen solutions to induce gelation are well established in the literature. Recently, a report in the literature[1] showed a novel method to fabricate highly oriented electrochemically aligned collagen (ELAC) threads which have orders of magnitude greater packing density than collagen gels. The optimal PBS treatment conditions for induction of D-banding pattern in such dense and anisotropic collagen network are unknown. This study aimed to optimize PBS treatment of ELAC threads by investigating the effect of phosphate ion concentration (0.5×, 1×, 5× or 10×) and incubation time (3, 12 or 96 hours) on the mechanical strength and ultrastructural organization by monotonic mechanical testing, small angle X-ray scattering and transmission electron microscopy. ELAC threads incubated in water (No PBS) served as the control. ELAC threads incubated in 1× PBS showed significantly higher extensibility compared to 0.5× or 10× PBS along with the presence of D-banded patterns with a periodicity of 63.83 nm. Incubation of ELAC threads in 1× PBS for 96 hours resulted in significantly higher ultimate stress compared to 3 or 12 hours. However, these threads lacked D-banding pattern. TEM showed no significant differences in the microfibril diameter distribution of ELAC threads treated with or without PBS. This indicates that microfibrils lacked D-banding following electrochemical alignment and the subsequent PBS treatment induced D-banding by reorganization within microfibrils. It was concluded that incubation of aligned collagen in 1× PBS for 12 hours results in mechanically competent, D-banded ELAC threads which can be used for the regeneration of load bearing tissues such as tendons and ligaments.

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There are approximately 33 million injuries involving musculoskeletal tissues (including tendons and ligaments) every year in the United States. In certain cases the tendons and ligaments are damaged irreversibly and require replacements that possess the natural functional properties of these tissues. As a biomaterial, collagen has been a key ingredient in tissue engineering scaffolds. The application range of collagen in tissue engineering would be greatly broadened if the assembly process could be better controlled to facilitate the synthesis of dense, oriented tissue-like constructs. An electrochemical method has recently been developed in our laboratory to form highly oriented and densely packed collagen bundles with mechanical strength approaching that of tendons. However, there is limited information whether this electrochemically aligned collagen bundle (ELAC) presents advantages over randomly oriented bundles in terms of cell response. Therefore, the current study aimed to assess the biocompatibility of the collagen bundles in vitro, and compare tendon derived fibroblasts (TDFs) and bone marrow stromal cells (MSCs) in terms of their ability to populate and migrate on the single and braided ELAC bundles. The results indicated that the ELAC was not cytotoxic; both cell types were able to populate and migrate on the ELAC bundles more efficiently than that observed for random collagen bundles. The braided ELAC constructs were efficiently populated by both TDFs and MSCs in vitro. Therefore, both TDFs and MSCs can be used with the ELAC bundles for tissue engineering purposes.