Regenerative Engineering and Translational Medicine

Three-Dimensional Porous Trabecular Scaffold Exhibits Osteoconductive Behaviors In Vitro Abstract In the USA, approximately 500,000 bone grafting procedures are performed annually to treat injured or diseased bone. Autografts and allografts are the most common treatment options but can lead to adverse outcomes such as donor site morbidity and mechanical failure within 10 years. Due to this, tissue engineered replacements have emerged as a promising alternative to the biological options. In this study, we characterize an electrospun porous composite scaffold as a potential bone substitute. Various mineralization techniques including electrodeposition were explored to determine the optimal method to integrate mineral content throughout the scaffold. In vitro studies were performed to determine the biocompatibility and osteogenic potential of the nanofibrous scaffolds. The presence of hydroxyapatite (HAp) and brushite throughout the scaffold was confirmed using energy dispersive X-ray fluorescence, scanning electron microscopy, and ash weight analysis. The active flow of ions via electrodeposition mineralization led to a threefold increase in mineral content throughout the scaffold in comparison to static and flow mineralization. Additionally, a ten-layer scaffold was successfully mineralized and confirmed with an alizarin red assay. In vitro studies confirmed the mineralized scaffold was biocompatible with human bone marrow derived stromal cells. Additionally, bone marrow derived stromal cells seeded on the mineralized scaffold with embedded HAp expressed 30% more osteocalcin, a primary bone protein, than these cells seeded on non-mineralized scaffolds and only 9% less osteocalcin than mature pre-osteoblasts on tissue culture polystyrene. This work aims to confirm the potential of a biomimetic mineralized scaffold for full-thickness trabecular bone replacement. Lay Summary Bioengineered options for trabecular bone replacement should be porous for cellular infiltration and bioactive to promote the formation of new bone tissue. This work features techniques to increase mineralization within full-thickness porous nanofibrous scaffolds. In vitro studies elucidated the biocompatibility and osteogenic potential of the mineralized trabecular scaffold by promoting human bone marrow–derived stromal cell proliferation and primary bone protein expression. The end goal of this work is to combine this porous trabecular scaffold with a pre-vascularized cortical bone scaffold to yield a full-dimensional biomimetic bone construct with the ability to promote simultaneous multidifferentiation of stem cells in vivo.

Gelatin-Based Microribbon Hydrogels Guided Mesenchymal Stem Cells to Undergo Endochondral Ossification In Vivo with Bone-Mimicking Mechanical Strength Abstract Most stem cell–based bone tissue engineering strategies to date yield bone through direct bone formation, which mimics intramembranous ossification. However, bone injuries often affect long bones which are formed through endochondral ossification, involving an initial cartilage template formation followed by remodeling to form bones. There remains a critical need to develop scaffolds that enhance stem cell–based bone formation through endochondral ossification with bone-mimicking mechanical strength. Here we evaluated the potential of gelatin-based microribbons (μRBs) as macroporous scaffolds for enhancing human mesenchymal stem cell (MSC)–based bone formation through endochondral ossification. This material platform was compared with conventional gelatin hydrogels (HGs) as controls. MSCs were encapsulated in μRB or HG scaffolds, primed in chondrogenic medium in vitro for 2 weeks, and then implanted in a mouse subcutaneous model with no additional factors. μRB scaffolds supported fast cartilage deposition by MSCs, which was completely remodeled and replaced by mineralized bone. Impressively, the compressive moduli of MSC-seeded μRB scaffolds increased from 10 to 3224 kPa by week 11, a range that mimics native bone. In comparison, while HG supported endochondral ossification, the speed was much slower, with less matrix deposition and only a modest increase in compressive modulus to 269 kPa. These results validate gelatin μRBs as a promising scaffold for repairing long bone defects by guiding robust endochondral ossification. Lay Summary Natural bone development and healing occurs through two distinct pathways: intramembranous ossification and endochondral ossification. Most bone injuries affect long bones, which are formed through endochondral ossification, involving an initial cartilage template formation followed by remodeling to form bones. However, scaffolds that can guide stem cell–based bone formation through endochondral ossification with bone-mimicking mechanical strength remain lacking. Here we report that macroporous gelatin-based microribbons (μRBs) accelerate endochondral ossification by human mesenchymal stem cells (MSCs) in vivo using a mouse subcutaneous model. Impressively, the mechanical properties of MSC-seeded μRB scaffolds increased over 300-fold over 11 weeks to bone-mimicking range, whereas conventional gelatin hydrogel controls reached less than 10% of the bone modulus. These results validate gelatin μRBs as a promising novel scaffold for repairing long bone defects by guiding robust endochondral ossification.

Comprehending the Unfolded Protein Response as a Conduit for Improved Mesenchymal Stem Cell-Based Therapeutics Abstract Endoplasmic reticulum (ER) is an intracellular organelle that contributes to efficacious protein synthesis, folding, assembly and transportation. When the normal functions of the ER are perturbed, ER stress is generated which in turn activates an unfolded protein response (UPR). UPR helps in combating this ER stress only at its threshold level, via the inhibition of protein synthesis. This is essentially mediated through increased chaperones and decreased proteins synthesis. Although when the cell undergoes continued ER-stress, UPR ultimately converges to cell-apoptosis, which leads to loss of MSCs viability. Therefore, a delineation into the molecular mechanisms of UPR would be an insightful attempt to curb the prominent loss of MSCs post-transplant. The regenerative potential of MSCs, along with their ubiquitous source of origin, offers vast opportunities with respect to the applications in diverse interdisciplinary sectors of research. But the prevalent loss of MSCs due to certain factors like reactive oxygen species (ROS), hypoxia-reperfusion (H/R), anoikis and inflammation, results in lack of persistence of transplanted MSCs. These hostile conditions contribute to an up surged endoplasmic reticulum stress. Hence, the current review aims at elucidating the mechanisms of the unfolded protein response and its regulation for improving MSCs viability. Lay Summary A major roadblock for successful use of adult stem cells in practice is their poor survivability once introduced in the patient’s body. The stem cells tend to die quickly due to stress caused by activation of the unfolded protein response (UPR) pathway. Hence if we can target this UPR pathway (de-activation), then the cells might be able to survive for longer period of time that can help in regeneration of the desired tissue in our body. Future Works Future work can focus on designing therapies that can specifically inhibit UPR activation in stem cells pre and post transplantation such that they are able to survive and help in the desired tissue regeneration inside our body.

Biomimetic Electroconductive Nanofibrous Matrices for Skeletal Muscle Regenerative Engineering Abstract Background The regeneration of the muscles of the rotator cuff represents a grand challenge in musculoskeletal regenerative engineering. Several types of matrices have been proposed for skeletal muscle regeneration. However, biomimetic matrices to promote muscle regeneration and mimic native muscle tissue have not been successfully engineered. Besides topographical cues, an electrical stimulus may serve as a critical cue to improve interactions between materials and cells in scenarios fostering muscle regeneration. In this in vitro study, we engineered a novel stimulus-responsive conductive nanocomposite matrix and studied its ability to regulate muscle cell adhesion, proliferation, and differentiation. Electroconductive nanocomposite matrices demonstrated tunable conductivity and biocompatibility. Under the optimum concentration of conductive material, the matrices facilitated muscle cell adhesion, proliferation, and differentiation. Importantly, aligned conductive fibrous matrices were effective in promoting myoblast differentiation by upregulation of myogenic markers. The results demonstrated a promising potential of aligned conductive fibrous matrices for skeletal muscle regenerative engineering. Lay Summary Around 40% of the human body mass consists of skeletal muscle. Musculoskeletal disorders such as muscle atrophy and fatty infiltration after rotator cuff injury lead to disability and pain and increase the rate of retear after rotator cuff surgery. The study showed the potential of novel engineered matrix to regenerate skeletal muscle by utilizing conductive material and nanofiber-based matrices. The incorporation of conductive material and aligned nanofibers as electrical and topographical cues significantly impacted cell viability and differentiation to support muscle regeneration. Future Work The study demonstrated that electroconductive nanocomposite matrix can favorably modulate myoblast proliferation and differentiation. Future study will investigate the in vivo efficacy of the engineered matrix using a rat rotator cuff tear model to understand the ability of the engineered matrix in reducing the fatty infiltration.

Correction to: Vision for Functionally Decorated and Molecularly Imprinted Polymers in Regenerative Engineering This paper was published without a complete acknowledgment.

Production and Characterization of Recombinant Collagen-Binding Resilin Nanocomposite for Regenerative Medicine Applications Abstract Development of mechanically stable and multifunctional biomaterials for sensing, repair, and regeneration applications is of great importance. Herein, we investigate the potential of recombinant resilin-like (Res) nanocomposite elastomer as a template biomaterial for regenerative devices such as adhesive bandages or films, electrospun fibers, screws, sutures, and drug delivery vehicles. Exon I (Rec1) from the native resilin gene of Drosophila (CG15920) was fused with collagen-binding domain (ColBD) from Clostridium histolyticum and expressed in Komagataella pastoris (formerly Pichia pastoris). The 100% binding of Resilin-ColBD (Res-ColBD) to collagen I was shown at a 1:1 ratio by mass. Atomic force microscopy results in force mode show a bimodal profile for the ColBD-binding interactions. Moreover, based on the force-volume map, Res-ColBD adhesion to collagen was statistically significantly higher than resilin without ColBD. Lay Summary Designing advanced biomaterials that will not only withstand the repetitive mechanical loading and flexibility of tissues but also retain biochemical and biophysical interactions remains challenging. The combination of physical, biological, and chemical cues is vital for disease regulation, healing, and ultimately complete regeneration of functional human tissues. Resilin is a super elastic and highly resilient natural protein with good biocompatibility but lacks specific biological and chemical cues. Therefore, resilin decorated with collagen I–binding domain is proposed as a functional nanocomposite template biomaterial. Collagen I is an ideal binding target, as it is the most abundant structural protein found in human body including scars that affect unwanted adhesion. Future Work Musculoskeletal-related injuries and disorders are the second largest cause of disabilities worldwide. Significant pain, neurological discomfort, limited mobility, and substantial financial burden are associated with these disorders. Thus, biocompatible materials comprised of resilin with collagen-binding domain, such as films adhesive bandages (films, fiber matts, or hydrogels), sutures, screws and rods, three-dimensional scaffolds, and delivery vehicles, will be designed and evaluated for multiple musculoskeletal-related regeneration applications.

Time-Dependent Addition of Neuronal and Schwann Cells Increase Myotube Viability and Length in an In Vitro Tri-culture Model of the Neuromuscular Junction Abstract The neuromuscular junction (NMJ) is a specialized chemical synapse between motor neurons and muscle fibers that enables voluntary movement of somatic muscle. Study of NMJ physiology and pathology has used various animal models and cell lines to recapitulate critical features of neuromuscular disease and increase our understanding of NMJ disruption. In vitro co-culture platforms have also been used to evaluate NMJ development using different combinations of motor neurons (MNs), glial Schwann Cells (SCs), skeletal muscle (SKM) cells, and biomaterials to advance translation of regenerative therapies. In this report, we use a compartmentalized, microfluidic platform to develop a novel, tri-culture model of the three cell types that comprise the NMJ: SKM cells, MNs, and SCs. Results illustrate the reproducible differentiation of SKM myotubes with increased viability and length following the time-dependent addition of neuronal and glial cells as introduced in vivo. The data points to Schwann Cells as key players to stabilize and maintain in vitro NMJ models that will aid development and testing of emerging therapies for neuromuscular dysfunction. Lay Summary Schwann cells (SCs) of the peripheral nervous system are essential to the development and function of the NMJ. Although recent evidence has revealed the significant roles of these glia in NMJ remodeling and regeneration, SCs have been largely overlooked in NMJ culture models. This project used a compartmentalized microfluidic platform to isolate and add each cellular component of the NMJ in a time-dependent manner to facilitate cellular interaction and stabilization. Results demonstrate that the time-dependent addition of SCs increased the viability and differentiated length of skeletal myotubes observed in vitro. Our contributions will aid the development of microfluidic, tri-culture models that utilize primary and stem-like cells to develop functional in vitro models with which to test and evaluate emerging regenerative NMJ therapies.

Sources of Variability in Manufacturing of Cell Therapeutics Abstract Cellular immunotherapies are expected to greatly impact the future therapeutic landscape. Like other innovative and transformative fields, the industry is currently refining to keep pace with available technology. As a result, developers are employing more controls to minimize variability, and suppliers are addressing industry needs by addressing supply issues and characterizing material attributes with the careful awareness to the impact of the cost of the therapies. Process and analytical technologies are continuing to develop along with the components that are needed for them. Lay Summary Future efforts will focus in closing the gap in standardization of processes and materials. Creation of standards can mitigate risks associated with the variability intrinsic to cell therapy products. Increased characterization of processes, methods, products, and materials will also address variability within the cell therapy field.

Identifying and Managing Sources of Variability in Cell Therapy Manufacturing and Clinical Trials Abstract Identifying and managing cell therapy variability can be a significant challenge for a company seeking to commercialize a new product. Failure to address this issue can lead to negative consequences such as delayed approval due to unsuccessful clinical investigations, or failed product lots that do not meet release criteria. Allogeneic cell therapies can be particularly prone to variability challenges due to the use of variable input material. In order to support the manufacturers of cell therapies, the FDA has identified two primary regulatory pathways (351 vs 361) that reflect the relative risk of the product. In this review, we will discuss criteria that separate the two potential regulatory pathways for cell therapy products in the USA. Also, we will discuss what aspects of manufacturing and clinical trial execution might introduce undesired variability that can derail the path towards licensure and commercialization, along with tools to minimize these potential sources of variability. ClinicalTrials.gov Identifier: NCT03347708 and NCT03955315 Lay Summary Therapies that utilize live cells as the active ingredient, known as cell therapies, are a promising approach to treating many diseases that cannot be addressed with traditional medicines. However, with this great potential comes specific challenges associated with cell therapy, including identifying the appropriate regulatory approval pathway, manufacturing it in a reproducible way, and successfully executing clinical trials. In this review, we describe potential sources of variability that can negatively impact the translation of a cell therapy, and ways to minimize those risks.

Neuropotency and Neurotherapeutic Potential of Human Umbilical Cord Stem Cell’s Secretome Abstract Background Recent studies have revealed that the therapeutic effect of mesenchymal stem cells (MSCs) is due to their secretome. Secretome is considered to be advantageous over cells, because of less chances of teratoma formation and no cell genome variability of donor and recipient. The current study aimed to screen the secretome of cord lining (CL) and Wharton jelly (WJ) of human umbilical cord stem cells and their functionality. Methods Explants culture of human CL and WJ were characterized for classical MSC markers, pluripotency for germ layer expression then studied for their neurosphere and neurogenesis ability. These stem cells were screened for the expression of trophic factors (BDNF/GDNF/CNTF/NT3/NT4/NGF/FGF1/FGF2/VEGF/HGF/IGF/EGF/PlGF/IFN gamma/TNF-α/IL-1b/IL-4/IL-10/IFN-α1/TGF-β). Cells were cultured under serum-free conditions for 48 h and the conditioned media (CM) were collected. Functional effect of CM was analyzed on SHSY5Y, U87MG, and endothelial cells for proliferation/differentiation/anti-inflammatory and pro-angiogenic effects respectively. Results CL and WJ cells showed positive for MSC markers and negative for hematopoietic lineage. Both stem cells exhibited highly proliferative phenotype with normal karyotype even after several passages. All the major growth factors were expressed at varying levels expect CNTF. Cells illustrated the expression of various inflammatory modulators except IL-10 and elucidated anti-inflammatory and pro-angiogenic effect. These cells formed neurosphere in vitro and upon neuronal induction, expressed mature neuronal markers. CM enhanced SHSY5Y cell proliferation and differentiation. Conclusion Easily accessible human umbilical cord stem’s secretome can have potential therapeutic effect in neurodegenerative disease. Lay Summary Cellular secretions from human umbilical cord stem cells have the potential property to exert neuroprotective effect. Hence, instead of using cells as the therapeutics, the cellular secretions can be potentially used to achieve clinical healing/repair of degenerative tissues of our nervous system.