Search

Injuries of the bone-to-tendon interface, such as rotator cuff and anterior cruciate ligament tears, are prevalent musculoskeletal injuries, yet effective methods for repair remain elusive. Tissue engineering approaches that use cells and biomaterials offer a promising potential solution for engineering the bone-tendon interface, but previous strategies require seeding multiple cell types and use of multiphasic scaffolds to achieve zonal-specific tissue phenotype. Furthermore, mimicking the aligned tissue morphology present in native bone-tendon interface in three-dimensional (3D) remains challenging. To facilitate clinical translation, engineering bone-tendon interface using a single cell source and one continuous scaffold with alignment cues would be more attractive but has not been achieved before. To address these unmet needs, in this study, we develop an aligned gelatin microribbon (μRB) hydrogel scaffold with hydroxyapatite nanoparticle (HA-np) gradient for guiding zonal-specific differentiation of human mesenchymal stem cell (hMSC) to mimic the bone-tendon interface. We demonstrate that aligned μRBs led to cell alignment in 3D, and HA gradient induced zonal-specific differentiation of mesenchymal stem cells that resemble the transition at the bone-tendon interface. Short chondrogenic priming before exposure to osteogenic factors further enhanced the mimicry of bone-cartilage-tendon transition with significantly improved tensile moduli of the resulting tissues. In summary, aligned gelatin μRBs with HA gradient coupled with optimized soluble factors may offer a promising strategy for engineering bone-tendon interface using a single cell source. Impact statement Our 3D macroporous microribbon hydrogel platform with alignment cues zonally integrated with hydroxyapatite nanoparticles enables differentiation across the bone-tendon interface within a continuous scaffold. While most interfacial scaffolds heretofore rely on composites and multilayer approaches, we present a continuous scaffold utilizing a single cell source. The synergy of niche cues with human mesenchymal stem cell (hMSC) culture leads to an over 45-fold enhancement in tensile modulus in culture. We further demonstrate that priming hMSCs towards the chondrogenic lineage can enhance the differential osteogenesis. Relying on a single cell source could enhance zone integration and scaffold integrity, along with practical benefits.

Bone defects caused by infection, tumor, trauma and so on remain difficult to treat clinically. Bone tissue engineering (BTE) has great application prospect in promoting bone defect repair. Polycaprolactone (PCL) is a commonly used material for creating BTE scaffolds. In addition, self-assembling peptides (SAPs) can function as the extracellular matrix and promote osteogenesis and angiogenesis. In the work, a PCL scaffold was constructed by 3D printing, then integrated with bone marrow mesenchymal stem cells (BMSCs) and SAPs. The research aimed to assess the bone repair ability of PCL/BMSC/SAP implants. BMSC proliferation in PCL/SAP scaffolds was assessed via Cell Counting Kit-8. In vitro osteogenesis of BMSCs cultured in PCL/SAP scaffolds was assessed by alkaline phosphatase staining and activity assays. Enzyme linked immunosorbent assays were also performed to detect the levels of osteogenic factors. The effects of BMSC-conditioned medium from 3D culture systems on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were assessed by scratch, transwell, and tube formation assays. After 8 weeks of in vivo transplantation, radiography and histology were used to evaluate bone regeneration, and immunohistochemistry staining was utilized to detect neovascularization. In vitro results demonstrated that PCL/SAP scaffolds promoted BMSC proliferation and osteogenesis compared to PCL scaffolds, and the PCL/BMSC/SAP conditional medium (CM) enhanced HUVEC migration and angiogenesis compared to the PCL/BMSC CM. In vivo results showed that, compared to the blank control, PCL, and PCL/BMSC groups, the PCL/BMSC/SAP group had significantly increased bone and blood vessel formation. Thus, the combination of BMSC-seeded 3D-printed PCL and SAPs can be an effective approach for treating bone defects.

Pharyngoesophageal defects can cause exposure to various bacterial flora and severe inflammation. We fabricated a biodegradable polycaprolactone (PCL) patch composed of both thin film and three-dimensional (3D) printed lattice, and then investigated the efficacy of pharyngoesophageal reconstruction by using 3D printed antibiotic-releasing PCL patches that inhibited early inflammation by sustained tetracycline (TCN) release from both thin PCL films and printed rods implanted in esophageal partial defects. PCL was 3D printed in lattice form on a presolution casted PCL thin film at ∼100 μm resolution. TCN was loaded onto the PCL-printed patches by 3D printing a mixture of TCN and PCL particles melted at 100°C. TCN exhibited sustained release

Vascularization is currently considered the biggest challenge in bone tissue engineering due to necrosis in the center of large scaffolds. We established a new expendable vascular bundle model to vascularize a three-dimensional printed channeled scaffold with and without bone morphogenetic protein-2 (BMP-2) for improved healing of large segmental bone defects. Bone formation and angiogenesis in an 8 mm critical-sized bone defect in the rat femur were significantly promoted by inserting a bundle consisting of the superficial epigastric artery and vein into the central channel of a large porous polycaprolactone scaffold. Vessels were observed sprouting from the vascular bundle inserted in the central tunnel. Although the regenerated bone volume in the group receiving the scaffold and vascular bundle was similar to that of the healthy femur, the rate of union of the group was not satisfactory (25% at 8 weeks). BMP-2 delivery was found to promote not only bone formation but also angiogenesis in the critical-sized bone defects. Both insertion of the vascular bundle alone and BMP-2 loading alone induced comparable levels of angiogenesis and when used in combination, significantly greater vascular volume was observed. These findings suggest a promising new modality of treatment in large bone defects. Level of Evidence: Therapeutic level I. IMPACT STATEMENT: Vascularization is currently the main challenge in bone tissue engineering. The combination of a vascular bundle and an osteoinductive three-dimensional printed graft significantly improved and accelerated bone regeneration and angiogenesis in critical-sized large bone defects, suggesting a promising new modality of treatment in large bone defects.

Articular cartilage lacks both a nutrient supply and progenitor cells. Once damaged, it has limited self-repair capability. Cartilage tissue engineering provides a promising strategy for regeneration, and the use of injectable hydrogels as scaffolds has recently attracted much attention. Silk fibroin (SF) is an advanced natural material used to construct injectable hydrogels that are nontoxic and can be used efficiently in crosslinking applications. The objective of the present work was to develop an injectable hydrogel using SF in a novel one-step ultrasonication crosslinking method. Gelation kinetics and the characteristics of ultrasonication-induced SF (US-SF) hydrogels were systematically evaluated. The cytocompatibility of US-SF hydrogels was evaluated using rabbit chondrocytes, the Cell Counting Kit-8 testing, and immunofluorescence staining. Furthermore, the

Bone marrow-derived mesenchymal stem cells (BMSCs) have the potential to form the mechanically responsive matrices of joint tissues, including the menisci of the knee joint. The purpose of this study is to assess BMSC's potential to engineer meniscus-like tissue relative to meniscus fibrochondrocytes (MFCs). MFCs were isolated from castoffs of partial meniscectomy from nonosteoarthritic knees. BMSCs were developed from bone marrow aspirates of the iliac crest. All cells were of human origin. Cells were cultured in type I collagen scaffolds under normoxia (21% O

Fabrication of multifunctional scaffolds with biomimicking physical and biological signals play an important role in enhancing tissue regeneration. Multifunctional features come from the composite scaffold with various bioactive molecules. However, simple, biocompatible, and controllable hybridization strategy is still lacking. In this study, we leverage naturally derived extracellular matrix (ECM) as chemically controllable hydrogel carrier to effectively load functional biomolecules. The use of ECM hydrogel takes advantage of both native functionality of ECM components and tunability of hydrogel in controlling release of loaded molecules. As a proof of concept, porous acellular bone scaffold was selected as the solid pristine scaffold to be composited with BMP-2 and VEGF, which are loaded by spinal cord-derived ECM (SC-ECM) hydrogel. Crosslinking degree of SC-ECM hydrogel is tuned by changing genipin concentration, which renders the control over release kinetics of BMP-2 and VEGF. The mechanical strength of scaffold maintained after hybridization and is not significantly decreased in wet condition. In vitro evaluations of scaffolds cocultured with osteoblasts and mesenchymal stem cells (MSCs) demonstrate the biocompatible and bioactive features resulting from the composite scaffolds. Evidenced by alkaline phosphatase test, immunofluorescence, and real-time polymerase chain reaction, differentiation of MSCs towards osteoblast lineage is significantly enhanced by composite scaffolds. Therefore, our strategy in fabricating composite scaffold enabled by biomolecule-loaded ECM hydrogel holds great promise in regenerating diverse tissue types by appropriate combinations of solid pristine scaffolds, ECM, and bioactive molecules. Impact statement We developed a bioactive molecule (e.g., growth factor, protein) loading method using extracellular matrix hydrogel as a carrier. It brings a new strategy to fabricate composite scaffolds with unique biofunctions.

Bioprinting of body tissues has gained great attention in recent years due to its unique advantages, including the creation of complex geometries and printing the patient-specific tissues with various drug and cell types. The most momentous part of the bioprinting process is bioink, defined as a mixture of living cells and biomaterials (especially hydrogels). Among different biomaterials, natural polymers are the best choices for hydrogel-based bioinks due to their intrinsic biocompatibility and minimal inflammatory response in body condition. Gelatin methacryloyl (GelMA) hydrogel is one of the high-potential hydrogel-based bioinks due to its easy synthesis with low cost, great biocompatibility, transparent structure that is useful for cell monitoring, photocrosslinkability, and cell viability. Furthermore, the potential of adjusting properties of GelMA due to the synthesis protocol makes it a suitable choice for soft or hard tissues. In this review, different methods for the bioprinting of GelMA-based bioinks, as well as various effective process parameters, are reviewed. Also, several solutions for challenges in the printing of GelMA-based bioinks are discussed, and applications of GelMA-based bioprinted tissues argued as well. Impact statement Bioprinting has been demonstrated as a promising and alternative approach for organ transplantation to develop various types of living tissue. Bioinks, with great biological characteristics similar to the host tissues and rheological/flow features, are the first requirements for the successful bioprinting approach. Gelatin methacryloyl (GelMA) hydrogel is one of the high-potential hydrogel-based bioinks. This review provides a comprehensive look at different methods for the bioprinting of GelMA-based bioinks and applications of GelMA-based bioprinted tissues for tissue repair.

Rebuilding the tooth-supporting tissues (periodontium) destroyed by periodontitis remains a clinical challenge. Periodontal ligament cells (PDLCs), multipotent cells within the periodontal ligament (PDL), differentiate and form new PDL and mineralized tissues (cementum and bone) during native tissue repair in response to specific extracellular matrix (ECM) cues. Thus, harnessing ECM cues to control PDLC activity ex vivo, and ultimately, to design a PDLC delivery vehicle for tissue regeneration is an important goal. In this study, poly(ethylene glycol) hydrogels were used as a synthetic PDL ECM to interrogate the roles of cell–matrix interactions and cell-mediated matrix remodeling in controlling PDLC activity. Results showed that PDLCs within matrix metalloproteinase (MMP)-degradable hydrogels expressed key PDL matrix genes and showed a six to eightfold increase in alkaline phosphatase (ALP) activity compared with PDLCs in nondegradable hydrogel controls. The increase in ALP activity, commonly considered an early marker of cementogenic/osteogenic differentiation, occurred independent of the presentation of the cell-binding ligand RGD or soluble media cues and remained elevated when inhibiting PDLC-matrix binding and intracellular tension. ALP activity was further increased in softer hydrogels regardless of degradability and was accompanied by an increase in PDLC volume. However, scaffolds that fostered PDLC ALP activity did not necessarily promote hydrogel ECM mineralization. Rather, matrix mineralization was greatest in stiffer, MMP-degradable hydrogels and required the presence of soluble media cues. These divergent outcomes illustrate the complexity of the PDLC response to ECM cues and the limitations of current scaffold materials. Nevertheless, key biomaterial design principles for controlling PDLC activity were identified for incorporation into scaffolds for periodontal tissue regeneration. IMPACT STATEMENT: Engineered scaffolds are an attractive approach for delivering periodontal ligament cells (PDLCs) to rebuild the tooth-supporting tissues. Replicating key extracellular matrix (ECM) cues within tissue engineered scaffolds may maximize PDLC potential. However, the identity of important ECM cues and how they can be harnessed to control PDLC activity is still unknown. In this study, matrix degradability, cell–matrix binding, and stiffness were varied using synthetic poly(ethylene glycol) hydrogels for three-dimensional PDLC culture. PDLCs exhibited dramatic and divergent responses to these cues, supporting further investigation of ECM-replicating scaffolds for control of PDLC behavior and periodontal tissue regeneration.

Positively charged oligo(poly(ethylene glycol) fumarate) (OPF+) hydrogel scaffolds, implanted into a complete transection spinal cord injury (SCI), facilitate a permissive regenerative environment and provide a platform for controlled observation of repair mechanisms. Axonal regeneration after SCI is critically dependent upon nutrients and oxygen from a newly formed blood supply. Our objective was to investigate fundamental characteristics of revascularization in association with the ingrowth of axons into hydrogel scaffolds, thereby defining spatial relationships between axons and the neovasculature. A novel combination of stereologic estimates and precision image analysis techniques quantitate neurovascular regeneration in rats. Multichannel hydrogel scaffolds containing Matrigel-only (MG), Schwann cells (SCs), or SCs with rapamycin-eluting poly(lactic co-glycolic acid) microspheres (RAPA) were implanted for 6 weeks following complete spinal cord transection. Image analysis of 72 scaffold channels identified a total of 2494 myelinated and 4173 unmyelinated axons at 10 μm circumferential intervals centered around 708 individual blood vessel profiles. Blood vessel number, density, volume, diameter, intervessel distances, total vessel surface and cross-sectional areas, and radial diffusion distances were compared. Axon number and density, blood vessel surface area, and vessel cross-sectional areas in the SC group exceeded that in the MG and RAPA groups. Individual axons were concentrated within a concentric radius of 200–250 μm from blood vessel walls, in Gaussian distributions, which identified a peak axonal number (Mean Peak Amplitude) corresponding to defined distances (Mean Peak Distance) from each vessel, the highest concentrations of axons were relatively excluded from a 25–30 μm zone immediately adjacent to the vessel, and from vessel distances >150 μm. Higher axonal densities correlated with smaller vessel cross-sectional areas. A statistical spatial algorithm was used to generate cumulative distribution F- and G-functions of axonal distribution in the reference channel space. Axons located around blood vessels were definitively organized as clusters and were not randomly distributed. A scoring system stratifies 5 direct measurements and 12 derivative parameters influencing regeneration outcomes. By providing methods to quantify the axonal-vessel relationships, these results may refine spinal cord tissue engineering strategies to optimize the regeneration of complete neurovascular bundles in their relevant spatial relationships after SCI. IMPACT STATEMENT: Vascular disruption and impaired neovascularization contribute critically to the poor regenerative capacity of the spinal cord after injury. In this study, hydrogel scaffolds provide a detailed model system to investigate the regeneration of spinal cord axons as they directly associate with individual blood vessels, using novel methods to define their spatial relationships and the physiologic implications of that organization. These results refine future tissue engineering strategies for spinal cord repair to optimize the re-development of complete neurovascular bundles in their relevant spatial architectures.

The present study sought to demonstrate the swelling behavior of hydrogel-microcarrier composite constructs to inform their use in controlled release and tissue engineering applications. In this study, gelatin methacrylate (GelMA) and GelMA-gelatin microparticle (GMP) composite constructs were three-dimensionally printed, and their swelling and degradation behavior was evaluated over time and as a function of the degree of crosslinking of included GMPs. GelMA-only constructs and composite constructs loaded with GMPs crosslinked with 10 mM (GMP-10) or 40 mM (GMP-40) glutaraldehyde were swollen in phosphate-buffered saline for up to 28 days to evaluate changes in swelling and polymer loss. In addition, scaffold reswelling capacity was evaluated under five successive drying–rehydration cycles. All printed materials demonstrated shear thinning behavior, with microparticle additives significantly increasing viscosity relative to the GelMA-only solution. Swelling results demonstrated that for GelMA/GMP-10 and GelMA/GMP-40 scaffolds, fold and volumetric swelling were statistically higher and lower, respectively, than for GelMA-only scaffolds after 28 days, and the volumetric swelling of GelMA and GelMA/GMP-40 scaffolds decreased over time. After 5 drying–rehydration cycles, GelMA scaffolds demonstrated higher fold swelling than both GMP groups while also showing lower volumetric swelling than GMP groups. Although statistical differences were not observed in the swelling of GMP-10 and GMP-40 particles alone, the interaction of GelMA/GMP demonstrated a significant effect on the swelling behaviors of composite scaffolds. These results demonstrate an example hydrogel-microcarrier composite system's swelling behavior and can inform the future use of such a composite system for controlled delivery of bioactive molecules in vitro and in vivo in tissue engineering applications. IMPACT STATEMENT: In this study, porous three-dimensional printed (3DP) hydrogel constructs with and without natural polymer microcarriers were fabricated to observe swelling and degradation behavior under continuous swelling and drying–rehydration cycle conditions. Inclusion of microcarriers with different crosslinking densities led to distinct swelling behaviors for each biomaterial ink tested. 3DP hydrogel and hydrogel-microcarrier composite scaffolds have been commonly used in tissue engineering for the delivery of biomolecules. This study demonstrates the swelling behavior of porous hydrogel and hydrogel-microcarrier scaffolds that may inform later use of such materials for controlled release applications in a variety of fields including materials development and tissue regeneration.

Traumatic joint injuries can result in significant cartilage defects, which can greatly increase the risk of osteoarthritis development. Due to the limited self-healing capacity of avascular cartilage, tissue engineering approaches are required for filling defects and promoting cartilage regeneration. Current approaches utilize invasive surgical procedures for extraction and implantation of autologous chondrocytes; therefore, injectable biomaterials have gained interest to minimize the risk of infection as well as patient pain and discomfort. In this study, we engineered biomimetic, hyaluronic acid (HA)-based cryogel scaffolds that possess shape-memory properties as they contract and regain their shape after syringe injection to noninvasively fill cartilage defects. The cryogels, fabricated with HA and glycidyl methacrylate at -20°C, resulted in an elastic, macroporous, and highly interconnected network that provided a conducive microenvironment for chondrocytes to remain viable and metabolically active after injection through a syringe needle. Chondrocytes seeded within cryogels and cultured for 15 days exhibited enhanced cell proliferation, metabolism, and production of cartilage extracellular matrix glycosaminoglycans compared with HA-based hydrogels. Furthermore, immunohistochemical staining revealed production of collagen type II from chondrocyte-seeded cryogels, indicating the maintenance of cell phenotype. These results demonstrate the potential of chondrocyte-seeded, HA-based, injectable cryogel scaffolds to promote regeneration of cartilage tissue for nonsurgically invasive defect repair. Impact statement Hyaluronic acid-based shape-memory cryogels provide a conducive microenvironment for chondrocyte adhesion, proliferation, and matrix biosynthesis for use in repair of cartilage defects. Due to their sponge-like elastic properties, cryogels can fully recover their original shape back after injection while not impacting metabolism or viability of encapsulated cells. Clinically, they provide an opportunity for filling focal cartilage defects by using a single, minimally invasive injection of a cell encapsulating biocompatible three-dimensional scaffold that can return to its original structure to fit the defect geometry and enable matrix regeneration.

The larynx is a fairly complex organ comprised of different muscles, cartilages, mucosal membrane, and nerves. Larynx cancer is generally the most common type of head and neck cancer. Treatment options are limited in patients with total or partial laryngectomy. Tissue-engineered organs have shown to be a promising alternative treatment for patients with laryngectomy. In this report we present an alternative and simple procedure to construct a whole pig larynx scaffold consisting of complete acellular structures of integrated muscle and cartilage. Larynges were decellularized (DC) using perfusion-agitation with detergents coupled with ultrasonication. DC larynges were then characterized to investigate the extracellular matrix (ECM) proteins, residual DNA, angiogenic growth factors, and morphological and ultrastructural changes to ECM fibers. After 17 decellularization cycles, no cells were observed in all areas of the larynx as confirmed by hematoxylin and eosin and DAPI (4',6-diamidino-2-phenylindole) staining. However, DC structures of dense thyroid and cricoid cartilage showed remnants of cells. All structures of DC larynges (epiglottis [

Introduction: For the regeneration of large volume tissue defects, the interaction between angiogenesis and osteogenesis is a crucial prerequisite. The surgically induced angiogenesis by means of a...

The basic concepts from the fields of biology and engineering are integrated into tissue engineering to develop constructs for the repair of damaged and/or absent tissues, respectively. The field has grown substantially over the past two decades, with particular interest in bone tissue engineering (BTE). Clinically, there are circumstances in which the quantity of bone that is necessary to restore form and function either exceeds the patient's healing capacity or bone's intrinsic regenerative capabilities. Vascularized osseous or osteocutaneous free flaps are the standard of care with autologous bone remaining the gold standard, but is commonly associated with donor site morbidity, graft resorption, increased operating time, and cost. Regardless of the size of a craniofacial defect, from trauma, pathology, and osteonecrosis, surgeons and engineers involved with reconstruction need to consider the complex three-dimensional (3D) geometry of the defect and its relationship to local structures. Three-dimensional printing has garnered significant attention and presents opportunities to use craniofacial BTE as a technology that offers a personalized approach to bony reconstruction. Clinicians and engineers are able to work together to produce patient-specific space-maintaining scaffolds tailored to site-specific defects, which are osteogenic, osseoconductive, osseoinductive, encourage angiogenesis/vasculogenesis, and mechanically stable upon implantation to prevent immediate failure. In this work, we review biological and engineering principles important in applying 3D printing technology to BTE for craniofacial reconstruction as well as present recent translational advancements in 3D printed bioactive ceramic scaffold technology. IMPACT STATEMENT: Surgical reconstruction for extensive bone defects has evolved over the last 20 years toward a more customized treatment approach which fulfill functional outcomes. Additionally, the merger of surgical and microvascular principles has given rise to custom tailored patient-specific free tissue flaps which reconstruct bony maxillofacial defects while rebuilding lining, soft tissue mass, and facial subunits—all of which are key to achieving outcomes that approach normalcy. The contemporary techniques for complex boney defect reconstruction remain constrained: autologous bone transfer is complicated by limited bone stock and shape, donor site morbidity, surgical site infection, delayed healing, long operative times, and cost. Due to the shortcomings associated with autologous bone, advances in bone tissue engineering (BTE), such as 3D printing for patient and site-specific devices, have sought to restore bone deficiencies using customizable devices (scaffolds).

Guided bone regeneration refers to the process in which bone defects could be regenerated by facilitated healing through the use of membranes, potentially with the delivery of osteoinductive molecules, however, the regeneration often failed due to inflammation during bone formation. In this study, we developed a membrane immobilized with lactoferrin to modulate both bone regeneration and inflammatory responses. Lactoferrin was immobilized on electrospun nanofibers (LF50) by exploiting an adhesive polydopamine coating method. When human adipose-derived stem cells (hADSCs) were seeded onto the nanofibers, the LF50 significantly increased the osteogenic differentiation. For example, the gene expression of osteopontin was 6.9 ± 2.3 times greater in the cells on LF50 than the cells on unmodified nanofibers without lactoferrin. In addition, the gene expression of tumor necrosis factor-alpha (

For patients with large, full-thickness burn wounds, sufficient donor sites for autografting are not available, and thus, alternate strategies must be used to close these wounds. Cultured epithelial autografts (CEAs) can aid in closing these wounds but are often associated with slow deposition of basement membrane proteins, leading to blistering and graft loss. Rete ridges and dermal papillae present at the dermal-epidermal junction (DEJ) play a key role in epidermal adhesion and skin homeostasis. Promoting the development of an interdigitated DEJ may enhance basement membrane protein deposition and provide enhanced physical interlock of the epidermis and dermis. To develop a dermal template with stable dermal papillae, an electrospun collagen scaffold was seeded with human dermal fibroblasts. Ridged topographies were patterned into the cell-seeded dermal template using laser ablation, creating wide and shallow (ActiveFX) or narrow and deep (DeepFX) wells. Micropatterned or flat (control) dermal templates were combined with CEAs immediately before grafting to full-thickness excisional wounds on immunodeficient mice. CEAs grafted in conjunction with ridged templates showed rete ridge formation at 2 weeks after grafting and led to increased epidermal thickness, proliferation, and stemness compared to templates with a flat DEJ. As this technology is further developed, the dermal papilla-containing dermal templates may be utilized in combination with CEAs to improve adhesion and clinical function. Impact statement Cultured epithelial autografts (CEAs) serve as an adjunct to conventional split-thickness autograft in patients with very large burns, but they are susceptible to blistering that can reduce engraftment. Blistering results, in part, from relatively slow basement membrane deposition after grafting. This study demonstrates that basement membrane deposition and rete ridge formation are enhanced by combination of CEAs with a micropatterned, cell-seeded dermal template. These findings may lead to improved treatment and increased survival in patients with very large burns.

Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts. Impact Statement For patients undergoing laryngectomy, no reconstruction methods are available to restore the complex functions of the human larynx. The first promising preclinical results have been achieved with the use of biological scaffolds fabricated from decellularized tissue. However, the complexity of laryngeal tissue composition remains a hurdle to create functional viable grafts, since previously each cell type requires tailored culture conditions. In this study, we report the de novo formation of three humanized laryngeal tissue compartments under one uniform culture condition, a possible keystone in creating vital composite tissue grafts for laryngeal regeneration.

Bone defects are common and, in many cases, challenging to treat. Tissue engineering is an interdisciplinary approach with promising potential for treating bone defects. Within tissue engineering, three-dimensional (3D) printing strategies have emerged as potent tools for scaffold fabrication. However, reproducibility and quality control are critical aspects limiting the translation of 3D printed scaffolds to clinical use, which remain to be addressed. To elucidate the factors that yield to the generation of defects in bioprinting and to achieve reproducible biomaterial printing, the objective of this article is to frame a systematic approach for optimizing and validating 3D printing of poly(caprolactone) (PCL)-hydroxyapatite (HAp) composite scaffolds. We delineate the effect of PCL-to-HAp ratio, print velocity, print temperature, and extrusion pressure on the architectural and mechanical properties of the 3D printed scaffold. Furthermore, we present an in situ image-based monitoring approach to quantify key quality-related aspects of constructs, such as the ability to deposit material consistently and print elementary shapes with fewer flaws. Our results show that small defects generated during the printing process have a significant role in lowering the mechanical properties of 3D printed polymeric scaffolds. In addition, the in vitro osteoinductivity of the fabricated scaffolds is demonstrated. IMPACT STATEMENT: Identifying quality control measures is essential in the translation of three-dimensional (3D) printed scaffolds into clinical practice. In this article, we highlighted the importance of selected printing parameters on the quality of the 3D printed scaffolds. We also demonstrated that flaws, such as voids, significantly lower the mechanical properties (compressive modulus) of 3D printed polymeric scaffolds.

Cell replacement therapy is a promising treatment strategy for Parkinson's disease (PD); however, the poor survival rate of transplanted neurons is a critical barrier to functional recovery. In this study, we used self-assembling peptide nanofiber scaffolds (SAPNS) based on the peptide RADA16-I to support the in vitro maturation and in vivo post-transplantation survival of encapsulated human dopaminergic (DA) neurons derived from induced pluripotent stem cells. Neurons encapsulated within the SAPNS expressed mature neuronal and midbrain DA markers and demonstrated in vitro functional activity similar to neurons cultured in two dimensions. A microfluidic droplet generation method was used to encapsulate cells within monodisperse SAPNS microspheres, which were subsequently used to transplant adherent, functional networks of DA neurons into the striatum of a 6-hydroxydopamine-lesioned PD mouse model. SAPNS microspheres significantly increased the in vivo survival of encapsulated neurons compared with neurons transplanted in suspension, and they enabled significant recovery in motor function compared with control lesioned mice using approximately an order of magnitude fewer neurons than have been previously needed to demonstrate behavioral recovery. These results indicate that such biomaterial scaffolds can be used as neuronal transplantation vehicles to successfully improve the outcome of cell replacement therapies for PD. IMPACT STATEMENT: Transplantation of dopaminergic (DA) neurons holds potential as a treatment for Parkinson's disease (PD), but low survival rates of transplanted neurons is a barrier to successfully improving motor function. In this study, we used hydrogel scaffolds to transplant DA neurons into PD model mice. The hydrogel scaffolds enhanced survival of the transplanted neurons compared with neurons that were transplanted in a conventional manner, and they also improved recovery of motor function by using significantly fewer neurons than have typically been transplanted to see functional benefits. This cell transplantation technology has the capability to improve the outcome of neuron transplantation therapies.

Volumetric muscle loss (VML) injuries, by definition, exceed the endogenous repair capacity of skeletal muscle resulting in permanent structural and functional deficits. VML injuries present a significant burden for both civilian and military medicine. Despite progress, there is still considerable room for therapeutic improvement. In this regard, tissue-engineered constructs show promise for VML repair, as they provide an opportunity to introduce both scaffolding and cellular components. We have pioneered the development of a tissue-engineered muscle repair (TEMR) technology created by seeding muscle progenitor cells onto a porcine-derived bladder acellular matrix followed by cyclic stretch preconditioning before implantation. Our work to date has demonstrated significant functional repair (60–90% functional recovery) in progressively larger rodent models of VML injury following TEMR implantation. Notwithstanding this success, TEMR implantation in cylindrically shaped VML injuries in the tibialis anterior (TA) muscle was associated with more variable functional outcomes than has been observed in sheet-like muscles such as the latissimus dorsi. In fact, previous observations documented a dichotomy of responses following TEMR implantation in a rodent TA VML injury model; with an ≈61% functional improvement observed in fewer than half (46%) of TEMR-implanted animals at 12 weeks postinjury. This current study builds directly from those observations as we modified the geometry of both the VML injury and the TEMR construct to determine if improved matching of the implanted TEMR construct to the surgically created VML injury resulted in increased functional recovery posttreatment. Following these modifications, we observed a comparable degree of functional improvement in a larger proportion of animals (≈67%) that was durable up to 24 weeks post-TEMR implantation. Moreover, in ≈25% of all TEMR-implanted animals, functional recovery was virtually complete (TEMR max responders), and furthermore, the functional recovery in all 67% of responding animals was accompanied by the presence of native-like muscle properties within the repaired TA muscle, including fiber cross-sectional area, fiber type, vascularization, and innervation. This study emphasizes the importance of tuning the application of tissue engineering technology platforms to the specific requirements of diverse VML injuries to improve functional outcomes. IMPACT STATEMENT: This report confirms and extends previous observations with our implantable tissue-engineered technology platform for repair of volumetric muscle loss (VML) injuries. Based on our prior work, we addressed factors hypothesized to be responsible for significant outcome variability following treatment of VML injuries in a rat tibialis anterior model. Through customization of the muscle repair technology to a specific VML injury, we were able to significantly increase the frequency at which functional recovery occurred, and furthermore, demonstrate durability out to 6 months. In addition, the enhanced biomimetic qualities of repaired muscle tissue were associated with the most robust functional outcomes.

Mineralized polymeric cryogels with interconnective macroporous structure have demonstrated their potential as promising scaffolding material in bone tissue engineering. However, their capability in inducing osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and osteogenesis in vivo has not been explored yet. In this work, the roles of the mineralized cryogel on osteogenesis are systematically studied. Mineralized macroporous poly(ethylene glycol)-co-2-hydroxyethyl methacrylate cryogel promotes osteogenic differentiation of rat MSCs, particularly in upregulating the activity of alkaline phosphatase (ALP, ∼5.7-folds) and expression of related osteogenic gene markers (ALP ∼16-folds, osteocalcin ∼133-folds) at 14 days. In vivo implantation reveals that mineralized cryogels could promote fast osteogenesis and angiogenesis in critical-sized cranial bone defect of a Sprague-Dawley rat model in 4 weeks. The adsorption, entrapment, and concentration of osteogenic growth factors (bone morphogenetic protein 2) and angiogenesis growth factor (vascular endothelial growth factor [VEGF]) in the matrices in vivo may possibly participate in the process of osteogenesis and angiogenesis. Notably, the adsorption of larger amount of VEGF in nonmineralized cryogels facilitates obvious angiogenesis and comparable osteogenesis in bone defect in 8 weeks. Graphical abstract [Figure: see text] Impact Statement The current work reported the fabrication and characterization of a biomimicking mineralized polymeric cryogel as scaffolding material in bone regeneration. In addition to its three dimensional porous structure and the osteogenic potential, this biomimicking scaffold was also found to enhance the adsorption of biochemical cues, which in turn greatly promoted the angiogenesis as well as the tissue regeneration.

Low back pain caused by degenerative disc disease affects many people worldwide and brings huge economical burden. Thus, attentions have focused on annulus fibrosus (AF) tissue engineering for treatment of intervertebral disc degeneration. To engineer a functional replacement for the AF, it is important to fabricate scaffolds that mimic the structural and mechanical properties of native tissue. AF-derived stem cells are promising seed cells for AF tissue engineering due to their tissue specificity. In the present study, decellularized AF matrix (DAFM)/chitosan hybrid hydrogels were fabricated using genipin as a crosslinker. AF stem cells were cultured on hydrogel scaffolds with or without basic fibroblast growth factor (bFGF), and cell proliferation, morphology, gene expression, and AF tissue synthesis were examined. Overall, more collagen-I, collagen-II, and aggrecan were secreted by AF stem cells grown on hydrogels with bFGF compared to those without. These results support the application of DAFM/chitosan hybrid hydrogels as an appropriate candidate for AF tissue engineering. Furthermore, incorporation of bFGF into hydrogels promoted AF-related tissue synthesis. Impact Statement The investigation of annulus fibrosus (AF)-related tissue secretion and gene expression in extracellular matrix (ECM) of AF-derived stem cells (AFSCs) provided theoretical and practical basis for the choice of scaffold materials and growth factors for AF tissue engineering. The innovations of the present work are obvious. First, AFSCs were used because they are more easily differentiated into AF cells, thereby producing more AF-related ECM. Second, the decellularized AF matrix (DAFM) was derived from native AF tissue, but had reduced immunogenicity after decellularization. Furthermore, the DAFM structure mimicked the fibrous network of actual AF tissue, which was advantageous to AFSC adhesion and growth. Third, basic fibroblast growth factor was successfully incorporated into the DAFM, showed gradual sustained release, and effectively promoted production of AF tissue ECM factors collagen-I, collagen-II, aggrecan, and glycosaminoglycan.

Electrospinning is an attractive method of fabricating nanofibers that replicate the ultrastructure of the human meniscus. However, it is challenging to approximate the mechanical properties of meniscal tissue while maintaining the biocompatibility of collagen fibers. Our objective was to determine if functionalizing polylactic acid (PLA) nanofibers with collagen would enhance their biocompatibility. We therefore used coaxial electrospinning to generate core–shell nanofibers with a core of PLA for mechanical strength and a shell of collagen to enhance cell attachment and matrix synthesis. We characterized the nanostructure of the engineered scaffolds and measured the hydrophilic and mechanical properties. We assessed the performance of human meniscal cells seeded on coaxial electrospun scaffolds to produce meniscal tissue by gene expression and histology. Finally, we investigated whether these cell-seeded scaffolds could repair surgical tears created ex vivo in avascular meniscal explants. Histology, immunohistochemistry, and mechanical testing of ex vivo repair provided evidence of neotissue that was significantly better integrated with the native tissue than with the acellular coaxial electrospun scaffolds. Human meniscal cell-seeded coaxial electrospun scaffolds may have potential in enhancing repair of avascular meniscus tears. IMPACT STATEMENT: The success of any tissue-engineered meniscus graft relies on its ability to mimic native three-dimensional microstructure, support cell growth, produce tissue-specific matrix, and enhance graft integration into the repair site. Polylactic acid scaffolds possess the desired mechanical properties, whereas collagen scaffolds induce better cell attachment and enhanced tissue regeneration. We therefore fabricated nanofibrous scaffolds that combined the properties of two biomaterials. These novel coaxial scaffolds more closely emulated the structure, mechanical properties, and biochemical composition of native meniscal tissue. Our findings of meniscogenic tissue generation and integration in meniscus defects have the potential to be translated to clinical use.

Losing one's ability to speak, because of tissue deficiency at the vocal fold (VF), leads to serious impairment in the quality of life. Until now, there is no successful approach for regenerating the VF. The aim of this study was to show the advantage of magnetic nanoparticles in the generation of scaffold-free three-dimensional (3D) VF cell constructs by magnetic tissue engineering (MTE). Rabbit VF fibroblasts were used to establish MTE: after cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs), cells can be controlled with a magnetic field thereby forming solid 3D cell structures. To transfer this method into human cells, SPIONs were adapted accordingly and tested for their influence on human VF (hVF) cells and for their ability to perform MTE with hVF cells. Of interest, the cell number and the magnet's shape influence the form of the rabbit VF cell construct. After successful characterization of hVF cells, biocompatibility analyses revealed no significant influence of SPIONs on them, thus 3D hVF cell constructs could be successfully generated by MTE. These basic results are important to develop MTE as an innovative method to regenerate functional VFs. We expect that in vivo studies, including MTE as an elegant, far-field controlled and touchless technology, will translate MTE VF bioconstructs into reconstructive laryngeal medicine. Impact Statement This study aims at nanotechnology for regenerative medicine by magnetic tissue engineering (MTE). New approaches for vocal fold (VF) reconstruction are desperately needed. Superparamagnetic iron oxide nanoparticles offer innovative, scaffold-free potentials for tissue engineering: MTE. By using MTE we could generate functional multilayered human VF cell constructs, which can consequently be used to regenerate the voice in patients with VF injuries.

The use of biomaterials for circumferential esophageal repair is technically challenging in a rat model, and an optimal scaffold implantation technique with nutritional support is essential. The purpose of this study was to investigate the effects of three-dimensional printed esophageal grafts and bioreactor cultivation on muscle regeneration and reepithelialization from circumferential esophageal defects in a rat model. Here, we designed an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a two-layered tubular scaffold and mesenchymal stem cell-based bioreactor system. The graft was verified by the performance comparison with an omentum-cultured esophageal scaffold. We also applied a new surgical anastomosis technique and a thyroid gland flap over the implanted scaffold to improve graft survival. Although no regenerated mucosal layer was observed around the implants of the control group, histological examination of the regenerative esophagi along the scaffold revealed that the bioreactor system and omentum-cultured groups showed more than 80% of the mucosal regeneration without a fistula. The regenerated tissues showed that the integration of the esophageal scaffold and its native esophageal tissue was intact and were covered with layers of stratified squamous epithelium with several newly developed blood vessels. Therefore, this study describes a novel approach for circumferential esophageal reconstruction. Impact Statement In vivo functional esophageal reconstruction remains challenging due to anastomosis site leakage and necrosis of the implanted scaffold in a circumferential esophageal defect. Therefore, it is necessary to develop a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle without leakage of the esophageal anastomosis. In this study, we proposed an intriguing strategy that combines a mesenchymal stem cell-seeded tubular scaffold with a bioreactor system for esophageal reconstruction and introduced a new surgical anastomosis technique with the thyroid gland flap over the implanted scaffold to improve graft survival. We believe that this system should be a powerful platform for circumferential replacement of the esophagus in a rat model.

Large craniofacial bone defects remain a clinical challenge due to their complex shapes and large volumes. Stem cell-based technologies that deliver osteogenic stem cells have shown remarkable regenerative potential but are hampered by the need for extensive in vitro manipulation before implantation. To address this, we explored the bone forming potential of the clinically relevant stromal vascular fraction (SVF) cells obtained from human lipoaspirate. SVF cells can be isolated for acute use in the operating room and contain a subpopulation of adipose-derived stromal/stem cells (ASCs) that can develop mineralized tissue. ASCs can be purified from the more heterogeneous population of SVF cells via secondary and tertiary culture on tissue culture plastic. In this study, the relative potential for using SVF cells or passaged ASCs to induce robust bone regeneration was compared. Isogenic SVF and ASCs were suspended in fibrin hydrogels and seeded in three-dimensional-printed osteoinductive scaffolds of decellularized bone matrix and polycaprolactone. In vitro, both cell populations successfully mineralized the scaffold, demonstrating the robust bone formation properties of SVF. In murine critical-sized cranial defects, ASC-loaded scaffolds had greater (but not statistically significant) bone volume and bone coverage area than SVF-loaded scaffolds. However, both cell-laden interventions resulted in significantly greater bone healing than contralateral acellular controls. In conclusion, we observed substantial in vitro mineralization and robust in vivo bone regeneration in tissue-engineered bone grafts using both SVF and passaged ASCs. Impact Statement The inability to effectively regenerate bone within critical-sized craniofacial defects is a present clinical challenge and overcoming this limitation using tissue engineering strategies would significantly advance current treatment outcomes. The present study tests the feasibility of harvesting stem cells intraoperatively, combining them with three-dimensional (3D)-printed osteoinductive scaffolds and, without culturing in vitro, implanting them into the bone defect to stimulate regeneration. The data from this study demonstrated that SVF isolated from lipoaspirate and used in vivo with minimal processing could be combined with a 3D-printed bioactive material in a point-of-care approach to promote bone regeneration.

We investigated the efficacy of a muscle-stuffed vein (MSV) seeded with neural-transdifferentiated human mesenchymal stem cells as an alternative nerve conduit to repair a 15-mm sciatic nerve defect in athymic rats. Other rats received MSV conduit alone, commercial polyglycolic acid conduit (Neurotube®), reverse autograft, or were left untreated. Motor and sensory functions as well as nerve conductivity were evaluated for 12 weeks, after which the grafts were harvested for histological analyses. All rats in the treatment groups demonstrated a progressive increase in the mean Sciatic Functional Index (motor function) and nerve conduction amplitude (electrophysiological function) and showed positive withdrawal reflex (sensory function) by the 10th week of postimplantation. Autotomy, which is associated with neuropathic pain, was severe in rats treated with conduit without cells; there was mild or no autotomy in the rats of other groups. Histologically, harvested grafts from all except the untreated groups exhibited axonal regeneration with the presence of mature myelinated axons. In conclusion, treatment with MSV conduit is comparable to that of other treatment groups in supporting functional recovery following sciatic nerve injury; and the addition of cells in the conduit alleviates neuropathic pain. Impact Statement It is shown that pretreated muscle-stuffed vein conduit is comparable to that of commercial nerve conduit and autograft in supporting functional recovery following peripheral nerve injury. The addition of neural-differentiated mesenchymal stem cells in the conduit is shown to alleviate neuropathic pain.

An essential step toward commercializing engineered tissues is to scale-up and automate their production. This presents a challenge for self-assembled tissues that are fragile at early time points and difficult to handle using robotic systems. The goal of this study was to automate tissue-engineered blood vessel (TEBV) fabrication by creating a custom cell seeding and self-assembly system that is conducive to robotic manipulation, coupled with a robotic system to assemble smooth muscle cell ring units into tissue tubes. To generate self-assembled tissue ring units manually, cells are seeded at a high density into custom agarose wells that have center posts (2 mm inner diameter), around which cells aggregate and contract to form rings. Agarose is well-suited for cell seeding and ring self-assembly because it is noncell adhesive, can be autoclaved, and reproducibly cast to form wells using silicone templates. However, agarose gel is soft and makes reliable robotic manipulation challenging. To solve this problem, we designed a custom ring self-assembly plate utilizing polyetherimide (PEI) wells with MED610 three-dimensional-printed center posts and an MED610 well negative that allows the casting of individual, annular agarose cell-seeding troughs within the PEI plate. Rings cultured in the new plate system were morphologically similar to rings cultured in control agarose wells and had a slightly higher failure load. To automate tube assembly, we created a unique robotic punch system to push tissue rings out of the PEI-MED610 plate onto a stainless steel mandrel to enable tube fusion. Tubes fabricated by manual or robotic ring removal and placement demonstrated similar morphology after tube fusion, and the automated system substantially reduced the time required to assemble tubes. In summary, we developed a novel robotic assembly system to precisely manipulate self-assembled tissue rings and enable scale-up and automation of TEBV biofabrication. IMPACT STATEMENT: Self-assembled tissues have potential to serve both as implantable grafts and as tools for disease modeling and drug screening. For these applications, tissue production must ultimately be scaled-up and automated. Limited technologies exist for precisely manipulating self-assembled tissues, which are fragile early in culture. Here, we presented a method for automatically stacking self-assembled smooth muscle cell rings onto mandrels, using a custom-designed well plate and robotic punch system. Rings then fuse into tissue-engineered blood vessels (TEBVs). This is a critical step toward automating TEBV production that may be applied to other tubular tissues as well.

The goal of this study was to develop a scaffold for the generation of an artificial intestine that specifically mimics the architecture and biomechanical properties of the native small intestine, and to evaluate the scaffold in vitro and in vivo. Scaffolds mimicking the microarchitecture of native intestine were fabricated from poly(glycerol sebacate) (PGS) with a thickness of 647 μm (±241 μm) and villus height of 340 μm (±29.5 μm). The scaffolds showed excellent biological properties, as 71.4% (±7.2%) and 58.7% (±12.7%) mass remained after 5 weeks of in vitro exposure to control and digestive media, respectively. Tensile properties of the scaffolds approached those of native porcine intestine and scaffolds maintained their mechanical properties over 6 weeks based on rheometer measurements. Scaffolds accommodated intestinal epithelial stem cells and demonstrated maintenance of size and microarchitecture after 12 weeks of omental implantation in mice. There was an expected amount of inflammation, but less tissue infiltration and tissue formation than anticipated. In conclusion, we developed novel scaffolds using PGS that mimic the microarchitecture and mechanical properties of native intestine with promise for use in artificial intestine for individuals with short bowel syndrome. IMPACT STATEMENT: This study is significant because it demonstrates an attempt to design a scaffold specifically for small intestine using a novel fabrication method, resulting in an architecture that resembles intestinal villi. In addition, we use the versatile polymer poly(glycerol sebacate) (PGS) for artificial intestine, which has tunable mechanical and degradation properties that can be harnessed for further fine-tuning of scaffold design. Moreover, the utilization of PGS allows for future development of growth factor and drug delivery from the scaffolds to promote artificial intestine formation.