Biomanufacturing Organized Collagen-Based Microfibers as a Tissue ENgineered Device (TEND) for Tendon Regeneration

Approximately 800,000 surgical repairs are performed annually in the U.S. for debilitating injuries to ligaments and tendons of the foot, ankle, knee, wrist, elbow and shoulder, presenting a significant healthcare burden. To overcome current treatment shortcomings and advance the treatment of tendon and ligament injuries, we have developed a novel electrospun Tissue ENgineered Device (TEND), comprised of type I collagen and poly(D,L-lactide) (PDLLA) solubilized in a benign solvent, dimethyl sulfoxide (DMSO). TEND fiber alignment, diameter and porosity were engineered to enhance cell infiltration leading to promote tissue integration and functional remodeling while providing biomechanical stability. TEND rapidly adsorbs blood and platelet-rich-plasma (PRP), gradually releases growth factors over two weeks. TEND further supported cellular alignment and upregulation of tenogenic genes from clinically relevant human stem cells within three days of culture. TEND implanted in a rabbit Achilles tendon injury model showed new in situ tissue generation, maturation, and remodeling of dense, regularly oriented connective tissue in vivo. In all, TEND\u27s organized microfibers, biological fluid and cell compatibility, strength and biocompatiblility make significant progress towards clinically translating electrospun collagen-based medical devices for improving the clinical outcomes of tendon injuries

Comprehensive Collagen Crosslinking Comparison of Microfluidic Wet-Extruded Microfibers for Bioactive Surgical Suture Development

Collagen microfiber-based constructs have garnered considerable attention for ligament, tendon, and other soft tissue repairs, yet with limited clinical translation due to strength, biocompatibility, scalable manufacturing, and other challenges. Crosslinking collagen fibers improves mechanical properties; however, questions remain regarding optimal crosslinking chemistries, biocompatibility, biodegradation, long-term stability, and potential for biotextile assemble at scale, limiting their clinical usefulness. Here, we assessed over 50 different crosslinking chemistries on microfluidic wet-extruded collagen microfibers made with clinically relevant collagen to optimize collagen fibers as a biotextile yarn for suture or other medical device manufacture. The endogenous collagen crosslinker, glyoxal, provides extraordinary fiber ultimate tensile strength near 300MPa, and Young\u27s modulus of over 3GPa while retaining 50% of the initial load-bearing capacity through 6 months as hydrated. Glyoxal crosslinked collagen fibers further proved cytocompatible and biocompatible per ISO 10993-based testing, and further elicits a predominantly M2 macrophage response. Remarkably these strong collagen fibers are amenable to industrial braiding to form strong collagen fiber sutures. Collagen microfluidic wet extrusion with glyoxal crosslinking thus progress bioengineered, strong, and stable collagen microfibers significantly towards clinical use for potentially promoting efficient healing compared to existing suture materials. STATEMENT OF SIGNIFICANCE: Towards improving clinical outcomes for over 1 million ligament and tendon surgeries performed annually, we report an advanced microfluidic extrusion process for type I collagen microfiber manufacturing for biological suture and other biotextile manufacturing. This manuscript reports the most extensive wet-extruded collagen fiber crosslinking compendium published to date, providing a tremendous recourse to the field. Collagen fibers made with clinical-grade collagen and crosslinked with glyoxal, exhibit tensile strength and stability that surpasses all prior reports. This is the first report demonstrating that glyoxal, a native tissue crosslinker, has the extraordinary ability to produce strong, cytocompatible, and biocompatible collagen microfibers. These collagen microfibers are ideal for advanced research and clinical use as surgical suture or other tissue-engineered medical products for sports medicine, orthopedics, and other surgical indications

Microfluidic fiber spinning for 3D bioprinting: Harnessing microchannels to build macrotissues

Microfluidics is rapidly revolutionizing the scientific panorama, providing unmatched high-throughput platforms that find application in numerous areas of physics, chemistry, biology, and materials science. Recently, microfluidic chips have been proposed, in combination with bioactive materials, as promising tools for spinning cell-laden fibers with on-demand characteristics. However, cells encapsulated in filaments produced via microfluidic spinning technology are confined in a quasi-three-dimensional (3D) environment that fails to replicate the intricate 3D architecture of biological tissues. Thanks to the recent synergistic combination of microfluidic devices with 3D bioprinting technologies that enable the production of sophisticated microfibers serving as the backbone of 3D structures, a new age of tissue engineering is emerging. This review looks at how combining microfluidics with 3D printing is contributing to the biofabrication of relevant human substitutes and implants. This paper also describes the whole manufacturing process from the production of the microfluidic tool to the printing of tissue models, focusing on cutting-edge fabrication technologies and emphasizing the most noticeable achievements for microfluidic spinning technology. A theoretical insight for thixotropic hydrogels is also proposed to predict the fiber size and shear stress developing within microfluidic channels. The potential of using microfluidic chips as bio-printheads for multi-material and multi-cellular bioprinting is discussed, highlighting the challenges that microfluidic bioprinting still faces in advancing the field of biofabrication for tissue engineering and regenerative medicine purposes

Mimicking the Hierarchical Organization of Natural Collagen: Toward the Development of Ideal Scaffolding Material for Tissue Regeneration

Biological materials found in living organisms, many of which are proteins, feature a complex hierarchical organization. Type I collagen, a fibrous structural protein ubiquitous in the mammalian body, provides a striking example of such a hierarchical material, with peculiar architectural features ranging from the amino acid sequence at the nanoscale (primary structure) up to the assembly of fibrils (quaternary structure) and fibers, with lengths of the order of microns. Collagen plays a dominant role in maintaining the biological and structural integrity of various tissues and organs, such as bone, skin, tendons, blood vessels, and cartilage. Thus, "artificial" collagen-based fibrous assemblies, endowed with appropriate structural properties, represent ideal substrates for the development of devices for tissue engineering applications. In recent years, with the ultimate goal of developing three-dimensional scaffolds with optimal bioactivity able to promote both regeneration and functional recovery of a damaged tissue, numerous studies focused on the capability to finely modulate the scaffold architecture at the microscale and the nanoscale in order to closely mimic the hierarchical features of the extracellular matrix and, in particular, the natural patterning of collagen. All of these studies clearly show that the accurate characterization of the collagen structure at the submolecular and supramolecular levels is pivotal to the understanding of the relationships between the nanostructural/microstructural properties of the fabricated scaffold and its macroscopic performance. Several studies also demonstrate that the selected processing, including any crosslinking and/or sterilization treatments, can strongly affect the architecture of collagen at various length scales. The aim of this review is to highlight the most recent findings on the development of collagen-based scaffolds with optimized properties for tissue engineering. The optimization of the scaffolds is particularly related to the modulation of the collagen architecture, which, in turn, impacts on the achieved bioactivity

Effect of Mammalian Tissue Source on the Molecular and Macroscopic Characteristics of UV-Cured Type I Collagen Hydrogel Networks

The tissue source of type I collagen is critical to ensure scalability and regulation-friendly clinical translation of new medical device prototypes. However, the selection of a commercial source of collagen that fulfils both aforementioned requirements and is compliant with new manufacturing routes is challenging. This study investigates the effect that type I collagen extracted from three different mammalian tissues has on the molecular and macroscopic characteristics of a new UV-cured collagen hydrogel. Pepsin-solubilised bovine atelocollagen (BA) and pepsin-solubilised porcine atelocollagen (PA) were selected as commercially available raw materials associated with varying safety risks and compared with in-house acid-extracted type I collagen from rat tails (CRT). All raw materials displayed the typical dichroic and electrophoretic characteristics of type I collagen, while significantly decreased lysine content was measured on samples of PA. Following covalent functionalisation with 4-vinylbenzyl chloride (4VBC), BA and CRT products generated comparable UV-cured hydrogels with significantly increased averaged gel content (G ≥ 97 wt.%), while the porcine variants revealed the highest swelling ratio (SR = 2224 ± 242 wt.%) and an order of magnitude reduction in compression modulus (Ec = 6 ± 2 kPa). Collectively, these results support the use of bovine tissues as a chemically viable source of type I collagen for the realisation of UV-cured hydrogels with competitive mechanical properties and covalent network architectures

Nanoengineered Biomaterials for Cell and Therapeutic Delivery

Direct-write extrusion bioprinting, a form of additive manufacturing, is a useful technique to recapitulate anatomical complexity for tissue engineering applications. However, bioprinting has hit a bottleneck in progress due to the lack of available bioinks with high printability, mechanical strength, and biocompatibility. Here, we report a family of hydrogel-based bioinks for extrusion bioprinting from poly (ethylene glycol) (PEG) and two-dimensional (2D) nanoparticles. PEG, a non-fouling easily modifiable polymer, combined with biocompatible Laponite XLG nanoparticles (2D nanosilicates) to obtain shear-thinning hydrogel bioinks. Electrostatic interactions between nanoparticles and hydrogen-bonding between polymer and nanoparticles govern the flow behavior and printability of bioink. The evaluation of hydrogel bioink using flow sweeps, peak holds, and dynamic oscillatory rheology, suggest that minimum shear-thinning index of ~0.3, solution viscosities >1000 Pa·s, and 80% recovery within 30s are necessary for printing high fidelity constructs. Mechanically stiff 3D printed structures are obtained by covalently crosslinking polymeric chains using ultraviolet (UV) light. Modifications to the PEG system through inclusion of dithiothreitol linkage or combining with gelatin methacrylate are used to control matrix degradation, cell adhesion properties, and therapeutic release. We envision that PEG bioinks can be used to print complex, large-scale, cell-laden tissue constructs with high structural fidelity and mechanical stiffness for applications in custom bioprinted scaffolds and tissue engineered implants

Nanoengineered Biomaterials for Cell and Therapeutic Delivery

Direct-write extrusion bioprinting, a form of additive manufacturing, is a useful technique to recapitulate anatomical complexity for tissue engineering applications. However, bioprinting has hit a bottleneck in progress due to the lack of available bioinks with high printability, mechanical strength, and biocompatibility. Here, we report a family of hydrogel-based bioinks for extrusion bioprinting from poly (ethylene glycol) (PEG) and two-dimensional (2D) nanoparticles. PEG, a non-fouling easily modifiable polymer, combined with biocompatible Laponite XLG nanoparticles (2D nanosilicates) to obtain shear-thinning hydrogel bioinks. Electrostatic interactions between nanoparticles and hydrogen-bonding between polymer and nanoparticles govern the flow behavior and printability of bioink. The evaluation of hydrogel bioink using flow sweeps, peak holds, and dynamic oscillatory rheology, suggest that minimum shear-thinning index of ~0.3, solution viscosities >1000 Pa·s, and 80% recovery within 30s are necessary for printing high fidelity constructs. Mechanically stiff 3D printed structures are obtained by covalently crosslinking polymeric chains using ultraviolet (UV) light. Modifications to the PEG system through inclusion of dithiothreitol linkage or combining with gelatin methacrylate are used to control matrix degradation, cell adhesion properties, and therapeutic release. We envision that PEG bioinks can be used to print complex, large-scale, cell-laden tissue constructs with high structural fidelity and mechanical stiffness for applications in custom bioprinted scaffolds and tissue engineered implants