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It has been shown that subacromial bursa (SAB) harbors connective tissue progenitor cells. The purpose of this study was to evaluate the effects of implantation of SAB-derived cells (SBCs) suspended in a fibrin sealant bead and implantation of SAB tissue at rotator cuff repair site on biomechanical properties of the repair in a mouse (C57Bl/6) model of supraspinatus tendon (ST) detachment and repair.Part 1: Murine SAB tissue was harvested and cultured. Viability of SBCs suspended in 10 μL of fibrin sealant beads was confirmed in vitro and in vivo. Eighty mice underwent right ST detachment and repair augmented with either fibrin sealant bead (control group) or fibrin sealant bead with 100,000 SBCs (study group) applied at the repair site. Part 2: 120 mice underwent right ST detachment and repair and were randomized equally into 4 groups: (1) a tissue group, which received a piece of freshly harvested SAB tissue; (2) a cell group, which received SBCs suspended in fibrin sealant bead; (3) a fibrin sealant group, which received plain fibrin sealant bead without cells; and (4) a control group, which received nothing at the ST repair site. An equal number of mice in each group were killed at 2 and 4 weeks. Specimens underwent biomechanical testing to evaluate failure force (part 1 and 2) and histologic analysis of the repair site (part 1 only).Part 1: The mean failure force in the study group was significantly higher than controls at 2 and 4 weeks (3.25 ± 1.03 N vs. 2.43 ± 0.56 N, P = .01, and 4.08 ± 0.99 N vs. 3.02 ± 0.8 N, P = .004, respectively). Mean cell density of the ST at the repair site was significantly lower in the study group at 2 weeks than in controls (18,292.13 ± 1706.41 vs. 29,501.90 ± 3627.49, P = .001). Study group specimens had lower proteoglycan contents than controls, but this difference was not statistically significant. Part 2: There was no difference in failure force between cell and tissue groups at the 2- and 4-week time points (P = .994 and P = .603, respectively). There was no difference in failure force between fibrin sealant bead and control groups at the 2- and 4-week time points (P = .978 and P = .752, respectively).This study shows that the application of SBCs and SAB tissue at the rotator cuff repair site increases the strength of repair in a murine model of rotator cuff detachment and repair.

Approximately half of annual musculoskeletal injuries in the US involve tendon tears. The naturally hypocellular and hypovascular tendon environment makes tendons injury-prone and heal slowly. Tendon tissue engineering strategies often use biomimetic scaffolds combined with bioactive factors and/or cells to enhance healing. FDA-approved growth factors to promote tendon healing are lacking, which highlights the need for safe and effective bioactive factors. Our previous work evaluated insulin as a bioactive factor and identified an optimal dose to promote in vitro mesenchymal stem cell survival, division, and tenogenesis. The present work evaluates the ability of insulin-functionalized electrospun nanofiber matrices with or without mesenchymal stem cells to enhance tendon repair in a rat Achilles injury model. Electrospun nanofiber matrices were functionalized with insulin, cultured with or without mesenchymal stem cells, and sutured to transected Achilles tendons in rats. We analyzed rat tendons 4 and 8 weeks after surgery for the tendon morphology, collagen production, and mechanical properties. Bioactive insulin-functionalized fiber matrices with mesenchymal stem cells resulted in significantly increased collagen I and III at 4 and 8 weeks postsurgery. Additionally, these matrices supported highly aligned collagen fibrils in the regenerated tendon tissue at 8 weeks. However, treatment- and control-regenerated tissues had similar tensile properties at 8 weeks, which were less than that of the native Achilles tendon. Our preliminary results establish the benefits of insulin-functionalized fiber matrices in promoting higher levels of collagen synthesis and alignment needed for functional recovery of tendon repair.

Peripheral nerve injuries account for roughly 3% of all trauma patients with over 900,000 repair procedures annually in the US. Of all extremity peripheral nerve injuries, 51% require nerve repair with a transected gap. The current gold-standard treatment for peripheral nerve injuries, autograft repair, has several shortcomings. Engineered constructs are currently only suitable for short gaps or small diameter nerves. Here, we investigate novel nerve guidance conduits with aligned microchannel porosity that deliver sustained-release of neurogenic 4-aminopyridine (4-AP) for peripheral nerve regeneration in a critical-size (15 mm) rat sciatic nerve transection model. The results of functional walking track analysis, morphometric evaluations of myelin development, and histological assessments of various markers confirmed the equivalency of our drug-conduit with autograft controls. Repaired nerves showed formation of thick myelin, presence of S100 and neurofilament markers, and promising functional recovery. The conduit's aligned microchannel architecture may play a vital role in physically guiding axons for distal target reinnervation, while the sustained release of 4-AP may increase nerve conduction, and in turn synaptic neurotransmitter release and upregulation of critical Schwann cell neurotrophic factors. Overall, our nerve construct design facilitates efficient and efficacious peripheral nerve regeneration via a drug delivery system that is feasible for clinical applications., Graphical abstract Image 1, Highlights • Nerve guidance conduit platform with tunable scaffold properties for repair and regeneration of large-gap nerve injuries. • Sustained 4-aminopyridine release amplifies neurotrophic factor release by Schwann cells to promote axon regeneration. • Longitudinally aligned scaffold pores and controllable physicochemical properties provide guidance for axon regeneration. • Critical-size rat sciatic nerve defect healing both structurally and functionally resembled autograft control treatment. • Innovative and transformative scaffold technology imbued with structural and functional features for tissue regeneration. • Scaffold enable tailorable release profiles for small molecules proteins and electrical stimulation for tissue regeneration.

Background The prognosis of glioblastoma (GBM) remains dismal because therapeutic approaches have limited effectiveness. A new targeted treatment using MEK inhibitors, including trametinib, has been proposed to improve GBM therapy. Trametinib had a promising preclinical effect against several cancers, but its adaptive treatment resistance precluded its clinical translation in GBM. Previously, we have demonstrated that protein arginine methyltransferase 5 (PRMT5) is upregulated in GBM and its inhibition promotes apoptosis and senescence in differentiated and stem-like tumor cells, respectively. We tested whether inhibition of PRMT5 can enhance the efficacy of trametinib against GBM. Methods Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, ELISA, and western blot were analyzed. In vivo, NSG mice were intracranially implanted with PRMT5-intact and -depleted GBMNS, treated with trametinib by daily oral gavage, and observed for tumor progression and mice survival rate. Results PRMT5 depletion enhanced trametinib-induced cytotoxicity in GBMNS. PRMT5 knockdown significantly decreased trametinib-induced AKT and ERBB3 escape pathways. However, ERBB3 inhibition alone failed to block trametinib-induced AKT activity suggesting that the enhanced antitumor effect imparted by PRMT5 knockdown in trametinib-treated GBMNS resulted from AKT inhibition and not ERBB3 inhibition. In orthotopic murine xenograft models, PRMT5-depletion extended the survival of tumor-bearing mice, and combination with trametinib further increased survival. Conclusion Combined PRMT5/MEK inhibition synergistically inhibited GBM in animal models and is a promising strategy for GBM therapy.

Whereas synthetic biodegradable polymers have been successfully applied for the delivery of biologics in other tissues, the anatomical complexity, poor blood supply, and reduced clearance of degradation byproducts in the rotator cuff create unique design challenges for implantable biomaterials. Here, we investigated lower molecular weight poly-lactic acid co-epsilon-caprolactone (PLA-CL) formulations with varying molecular weight and film casting concentrations as potential matrices for the therapeutic delivery of biologics in the rotator cuff. Matrices were fabricated with target footprint dimensions to facilitate controlled and protected release of model biologic (Bovine Serum Albumin), and anatomically-unhindered implantation under the acromion in a rodent model of acute rotator cuff repair. The matrix obtained from the highest polymeric-film casting concentration showed a controlled release of model biologics payload. The tested matrices rapidly degraded during the initial 4 weeks due to preferential hydrolysis of the lactide-rich regions within the polymer, and subsequently maintained a stable molecular weight due to the emergence of highly-crystalline caprolactone-rich regions. pH evaluation in the interior of the matrix showed minimal change signifying lesser accumulation of acidic degradation byproducts than seen in other bulk-degrading polymers, and maintenance of conformational stability of the model biologic payload. The context-dependent biocompatibility evaluation in a rodent model of acute rotator cuff repair showed matrix remodeling without eliciting excessive inflammatory reaction and is anticipated to completely degrade within 6 months. The engineered PLA-CL matrices offer unique advantages in controlled and protected biologic delivery, non-toxic biodegradation, and biocompatibility overcoming several limitations of commonly-used biodegradable polyesters.

Despite progress, clinical translation of tissue engineering (TE) products/technologies is limited. A significant effort is underway to develop biomaterials and cells through a minimally modified process for clinical translation of TE products. Recently, bone marrow aspirate (BMA) was identified as an autologous source of cells for TE applications and is currently being tested in clinical therapies, but the isolation methods need improvement to avoid potential for contamination and increase progenitor cell yield. To address these issues, we reproducibly processed human peripheral blood (PB) and BMA to develop autologously derived biomaterials and cells. We demonstrated PB-derived biomaterial/gel cross-linking and fibrin gel formation with varied gelation times as well as biocompatibility through support of human bone marrow-derived stem cell survival and growth in vitro. Next, we established a plastic culture-free process that concentrates and increases the yield of CD146(+)/CD271(+) early mesenchymal progenitor cells in BMA (concentrated BMA [cBMA]). cBMA exhibited increased colony formation and multipotency (including chondrogenic differentiation) in vitro compared with standard BMA. PB-derived gels encapsulated with cBMA also demonstrated increased cell proliferation and enhanced mineralization when assessed for bone TE in vitro. This strategy can potentially be developed for use in any tissue regeneration application; however, bone regeneration was used as a test bed for this study. IMPACT STATEMENT: Tissue engineering (TE) is a rapidly growing field; however, clinical translation of TE products remains challenging. Autologously sourced bone marrow aspirate (BMA) and peripheral blood (PB) offer relative ease of harvesting and host compatibility and require minimal/no regulatory approvals. In this study, concentration of BMA led to the enrichment of early progenitor cells, which can potentially improve TE outcomes. Furthermore, concentrating BMA in combination with calcium-mediated, cross-linked fibrin gels derived from PB presented enhanced proliferation and osteogenic potential for a minimally modified bone TE strategy. These findings demonstrate the potential utilization of BMA in combination with PB for development of clinically translatable TE strategies for regeneration of different tissues.

Advent of additive manufacturing in biomedical field has nurtured fabrication of complex, customizable and reproducible orthopaedic implants. Layer-by-layer deposition of biodegradable polymer employed in development of porous orthopaedic screws promises gradual dissolution and complete metabolic resorption thereby overcoming the limitations of conventional metallic screws. In the present study, screws with different pore sizes (916 × 918 μm to 254 × 146 μm) were 3D printed at 200 μm layer height by varying printing parameters such as print speed, fill density and travel speed to augment the bone ingrowth. Micro-CT analysis and scanning electron micrographs of screws with 45% fill density confirmed porous interconnections (40.1%) and optimal pore size (259 × 207 × 200 μm) without compromising the mechanical strength (24.58 ± 1.36 MPa). Due to the open pore structure, the 3D printed screws showed increased weight gain due to the deposition of calcium when incubated in simulated body fluid. Osteoblast-like cells attached on screw and infiltrated into the pores over 14 days of in vitro culture. Further, the screws also supported greater human mesenchymal stem cell adhesion, proliferation and mineralized matrix synthesis over a period of 21 days in vitro culture as compared to non-porous screws. These porous screws showed significantly increased vascularization in a rat subcutaneous implantation as compared to control screws. Porous screws produced by additive manufacturing may promote better osteointegration due to enhanced mineralization and vascularization., Graphical abstract Image 1, Highlights • 3D printing of biodegradable porous orthopedic screws. • Tunable porosity and pore size. • Calcium deposition in in-vitro degradation enhance mineralization. • Enhanced proliferation and mineralization in bone marrow derived mesenchymal stem cells. • Neovascularisation in promoted in case of porous biodegradable screws.

A biocompatible and biodegradable scaffold with load-bearing ability is required to enhance the repair of bone defects by facilitating the attachment, and proliferation of cells, and vascularization during new bone formation. However, it is challenging to maintain the porosity and biodegradability, as well as mechanical properties (especially compressive strength), at the same time. Therefore, in the present work, a biodegradable composite structure of poly(caprolactone) (PCL) was designed using compression molding with varying amounts of poly(glycolic acid) (PGA) (25, 50, 75 wt%) and fixed amount (20 wt%) of beta tricalcium phosphate (beta TCP). It was hypothesized that the fabricated composite structure will develop porosity during the degradation of the PGA and that the corresponding decrease in mechanical properties will be compensated by new bone formation and ingrowth, in vivo. Accordingly, we have systematically studied the effects of sample composition on time-dependent dissolution and mechanical properties of the PGA/beta TCP scaffolds. The compressive strength increased up to ~92 MPa at 50% compression of the designed PCL-PGA samples. Furthermore, the dissolution rate, as well as weight loss, was observed to increase with an increase in the PGA amount in PCL. Based on the mechanical properties and dissolution data, it is concluded that the PCL-PGA scaffolds with beta TCP can be suitable candidates for bone tissue engineering applications, specifically for the reconstruction of bone defects, where strength and biodegradation are both important characteristics.

Electrical stimulation (ES) is predominantly used as a physical therapy modality to promote tissue healing and functional recovery. Research efforts in both laboratory and clinical settings have shown the beneficial effects of this technique for the repair and regeneration of damaged tissues, which include muscle, bone, skin, nerve, tendons, and ligaments. The collective findings of these studies suggest ES enhances cell proliferation, extracellular matrix (ECM) production, secretion of several cytokines, and vasculature development leading to better tissue regeneration in multiple tissues. However, there is still a gap in the clinical relevance for ES to better repair tissue interfaces, as ES applied clinically is ineffective on deeper tissue. The use of a conducting material can transmit the stimulation applied from skin electrodes to the desired tissue and lead to an increased function on the repair of that tissue. Ionically conductive (IC) polymeric scaffolds in conjunction with ES may provide solutions to utilize this approach effectively. Injectable IC formulations and their scaffolds may provide solutions for applying ES into difficult to reach tissue types to enable tissue repair and regeneration. A better understanding of ES-mediated cell differentiation and associated molecular mechanisms including the immune response will allow standardization of procedures applicable for the next generation of regenerative medicine. ES, along with the use of IC scaffolds is more than sufficient for use as a treatment option for single tissue healing and may fulfill a role in interfacing multiple tissue types during the repair process., Graphical abstract Image 1, Highlights • Electrical stimulation (ES) is used as a physical therapy modality to facilitate functional rehabilitation. • ES enhances cell proliferation, extracellular matrix (ECM) synthesis, cytokines production, and vasculature development in tissues. • Beneficial effects of ES in research and clinics is evident for muscle, bone, skin, nerve, tendons and ligaments. • Ionically conductive (IC) polymers carry charges due to counter flow of ions under ES. • ES in combination with IC polymeric structures may provide alternative means for tissue repair and regeneration.

Peripheral nerve injuries can be extremely debilitating, resulting in sensory and motor loss-of-function. Endogenous repair is limited to non-severe injuries in which transection of nerves necessitates surgical intervention. Traditional treatment approaches include the use of biological grafts and alternative engineering approaches have made progress. The current article serves as a comprehensive, in-depth perspective on peripheral nerve regeneration, particularly nerve guidance conduits and drug delivery strategies. A detailed background of peripheral nerve injury and repair pathology, and an in-depth look into augmented nerve regeneration, nerve guidance conduits, and drug delivery strategies provide a state-of-the-art perspective on the field.

Although bone tissue allografts and autografts aremoften used as a regenerative tissue during the bone healing, their availability, donor site morbidity, and immune response to grafted tissue are limiting factors their more common usage. Tissue engineered implants, such as acellular or cellular polymeric structures, can be an alternative solution. A variety of scaffold fabrication techniques including electrospinning, particulate leaching, particle sintering, and more recently 3D printing have been used to create scaffolds with interconnected pores and mechanical properties for tissue regeneration. Simply combining particle sintering and molecular self-assembly to create porous microstructures with imbued nanofibers to produce micronanostructures for tissue regeneration applications. Natural polymers like polysaccharides, proteins and peptides of plant or animal origin have gained significant attention due to their assured biocompatibility in tissue regeneration. However, majority of these polymers are water soluble and structures derived from them are in the form of hydrogels and require additional stabilization via cross-linking. For bone healing applications scaffolds are required to be strong, and support attachment, proliferation and differentiation of osteoprogenitors into osteoblasts. Our ongoing work utilizes plant polysaccharide cellulose derivatives and collagen to create mechanically stable and bioactive micronanostructured scaffold for bone tissue engineering. Scaffold microstructure is essentially solvent sintered cellulose acetate (CA) microspheres in the form of a negative template for trabecular bone with defined pore and mechanical properties. Collagen nanostructures are imbued into the 3D environment of CA scaffolds using collagen molecular self-assembly principles. The resultant CA-collagen micronanostructures provide the benefits of combined polymers and serve as an alternative material platform to many FDA approved polyesters. Our ongoing studies and published work confirm improved osteoprogenitor adhesion, proliferation, migration, differentiation, extracellular matrix (ECM) secretion in promoting bone healing. In this chapter we will provide a detailed protocol on the creation of micronanostructured CA-collagen scaffolds and their characterization for bone tissue engineering using human mesenchymal stem cells.

Silica biomaterials including Bioglass offer great biocompatibility and bioactivity but fail to provide pore and degradation features needed for tissue engineering. Herein we report on the synthesis and characterization of novel amorphous silica fiber matrices to overcome these limitations. Amorphous silica fibers were fused by sintering to produce porous matrices. The effects of sacrificial polymer additives such as polyvinyl alcohol (PVA) and cellulose fibers (CF) on the sintering process were also studied. The resulting matrices formed between sintering temperatures of 1,350-1,550 °C retained their fiber structures. The matrices presented pores in the range of 50-200 μm while higher sintering temperatures resulted in increased pore diameter. PVA addition to silica significantly reduced the pore diameter and porosity compared with silica matrices with or without the addition of CF. The PVA additive morphologically appeared to fuse the silica fibers to a greater extent and resulted in significantly higher compressive modulus and strength than the rest of the matrices synthesized. These matrices lost roughly 30% of their original mass in an

3D bioprinting has enabled the creation of biomimetic tissue constructs for regenerative medicine and in vitro model systems. Large-scale production of 3D structures at the micron-scale resolution is achieved through bioprinting using custom bioinks. Stability and 3D construct compliance play an important role in offering cells with biomechanical cues that regulate their behavior and enable in vivo implantation. Various crosslinking strategies are developed to stabilize the 3D printed structures and new methodologies are constantly being evaluated to overcome the limitations of the existing methods. Photo-crosslinking has emerged as a simple and elegant technique that offers precise control over the spatiotemporal gelation of bioinks during bioprinting. This article summarizes the use of photo-crosslinking agents and methodology towards optimizing 3D constructs for specific biomedical applications. The article also takes into account various bioinks and photo-crosslinkers in creating stable 3D printed structures that offer bioactivity with desirable physicochemical properties. The current challenges of 3D bioprinting and new directions that can advance the field in its wide applicability to create 3D tissue models to study diseases and organ transplantation are also summarized.

Complex craniofacial surgeries of damaged tissues have several limitations, which present complications and challenges when trying to replicate facial function and structure. Traditional treatment techniques have shown suitable nerve function regeneration with various drawbacks. As technology continues to advance, new methods have been explored in order to regenerate damaged nerves in an effort to more efficiently and effectively regain original function and structure. This article will summarize recent bioengineering strategies involving biodegradable composite scaffolds, bioactive factors, and external stimuli alone or in combination to support peripheral nerve regeneration. Particular emphasis is made on the contributions of growth factors and electrical stimulation on the regenerative process., Graphical abstract Image 1, Highlights • Craniofacial nerve repair surgeries have limitations and often result in insufficient restoration of facial function. • Nerve repair strategies for critical defects have often resulted in failure to re-establish sufficient nerve function. • Biochemical molecules promote tissue regeneration by differentiation of recruited cells to mature neuronal fates. • Electrical stimulation promotes regeneration of axons and provide signals for native cells to differentiate.

Correction for ‘Growing a backbone – functional biomaterials and structures for intervertebral disc (IVD) repair and regeneration: challenges, innovations, and future directions’ by Matthew D. Harmon et al., Biomater. Sci., 2020, 8, 1216–1239, DOI: 10.1039/C9BM01288E.

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

In this paper, we report on a novel biocompatible micromechanical bioreactor (actuator and sensor) designed for the in situ manipulation and characterization of live microtissues. The purpose of this study was to develop and validate an application-targeted sterile bioreactor that is accessible, inexpensive, adjustable, and easily fabricated. Our method relies on a simple polydimethylsiloxane (PDMS) molding technique for fabrication and is compatible with commonly-used laboratory equipment and materials. Our unique design includes a flexible thin membrane that allows for the transfer of an external actuation into the PDMS beam-based actuator and sensor placed inside a conventional 35 mm cell culture Petri dish. Through computational analysis followed by experimental testing, we demonstrated its functionality, accuracy, sensitivity, and tunable operating range. Through time-course testing, the actuator delivered strains of over 20% to biodegradable electrospun poly (D, L-lactide-co-glycolide) (PLGA) 85:15 non-aligned nanofibers (~91 &micro, m thick). At the same time, the sensor was able to characterize time-course changes in Young&rsquo, s modulus (down to 10&ndash, 150 kPa), induced by an application of isopropyl alcohol (IPA). Furthermore, the actuator delivered strains of up to 4% to PDMS monolayers (~30 &micro, m thick), simultaneously characterizing their elastic modulus up to ~2.2 MPa. The platform repeatedly applied dynamic (0.23 Hz) tensile stimuli to live Human Dermal Fibroblast (HDF) cells for 12 hours (h) and recorded the cellular reorientation towards two angle regimes, with averages of &minus, 58.85&deg, and +56.02&deg, The device biocompatibility with live cells was demonstrated for one week, with no signs of cytotoxicity. We can conclude that our PDMS bioreactor is advantageous for low-cost tissue/cell culture micromanipulation studies involving mechanical actuation and characterization. Our device eliminates the need for an expensive experimental setup for cell micromanipulation, increasing the ease of live-cell manipulation studies by providing an affordable way of conducting high-throughput experiments without the need to open the Petri dish, reducing manual handling, cross-contamination, supplies, and costs. The device design, material, and methods allow the user to define the operational range based on their targeted samples/application.

Back pain and associated maladies can account for an immense amount of healthcare cost and loss of productivity in the workplace. In particular, spine related injuries in the US affect upwards of 5.7 million people each year. The degenerative disc disease treatment almost always arises due to a clinical presentation of pain and/or discomfort. Preferred conservative treatment modalities include the use of non-steroidal anti-inflammatory medications, physical therapy, massage, acupuncture, chiropractic work, and dietary supplements like glucosamine and chondroitin. Artificial disc replacement, also known as total disc replacement, is a treatment alternative to spinal fusion. The goal of artificial disc prostheses is to replicate the normal biomechanics of the spine segment, thereby preventing further damage to neighboring sections. Artificial functional disc replacement through permanent metal and polymer-based components continues to evolve, but is far from recapitulating native disc structure and function, and suffers from the risk of unsuccessful tissue integration and device failure. Tissue engineering and regenerative medicine strategies combine novel material structures, bioactive factors and stem cells alone or in combination to repair and regenerate the IVD. These efforts are at very early stages and a more in-depth understanding of IVD metabolism and cellular environment will also lead to a clearer understanding of the native environment which the tissue engineering scaffold should mimic. The current review focusses on the strategies for a successful regenerative scaffold for IVD regeneration and the need for defining new materials, environments, and factors that are so finely tuned in the healthy human intervertebral disc in hopes of treating such a prevalent degenerative process.

Artificial nerve guidance conduits (NGCs) are being investigated as an alternative to autografts, since autografts are limited in supply. A polycaprolactone (PCL)-based spiral NGC with crosslinked laminin on aligned nanofibers was evaluated in vivo post a successful in vitro assessment. PC-12 cell assays confirmed that the aligned nanofibers functionalized with laminin were able to guide and enhance neurite outgrowth. In the rodent model, the data demonstrated that axons were able to regenerate across the critical nerve gap, when laminin was present. Without laminin, the spiral NGC with aligned nanofibers group resulted in a random cluster of extracellular matrix tissue following injuries. The reversed autograft group performed best, showing the most substantial improvement based on nerve histological assessment and gastrocnemius muscle measurement. Nevertheless, the recovery time was too short to obtain meaningful data for the motor functional assessments. A full motor recovery may take up to years. An interesting observation was noted in the crosslinked laminin group. Numerous new blood capillary-like structures were found around the regenerated nerve. Owing to recent studies, we hypothesized that new blood vessel formation could be one of the key factors to increase the successful rate of nerve regeneration in the current study. Overall, these findings indicated that the incorporation of laminin into polymeric nerve conduits is a promising strategy for enhancing peripheral nerve regeneration. However, the best combination of contact-guidance cues, haptotactic cues, and chemotactic cues have yet to be realized. The natural sequence of nerve regeneration should be studied more in-depth before modulating any strategies.

Soft tissue refers to those that support, connect, or surround the body’s organs. This includes muscles, ligaments, tendons, nerve, fat, fascia, and blood vessels. Soft tissues are susceptible to injuries, especially for athletes and the elderly population leading to an increase in the demand for surgeries. However, surgical intervention cannot provide full biological restoration of the tissues, resulting in scar formation rather than regeneration. Tissue engineering aims to develop tissue substitutes by employing engineered extracellular matrix (ECM), cells and factors alone or in combination to overcome limitations in tissue healing. It is expected that the demand for tissue-engineered products and approaches addresses clinically unmet needs in soft-tissue regeneration. The ultimate goal of tissue engineering is to design bioactive implanted scaffolds that mimic the native ECM of the target tissue structurally. These designed scaffolds provide the required mechanical and structural properties that aid the damaged tissue. Nanofiber matrices derived from natural and synthetic materials closely mimic the natural ECM structurally and compositionally. These matrices provide large surface area to volume ratio and adjustable pore dimeter and mechanical strength to enable the soft-tissue repair and subsequent soft-tissue regeneration. Therefore, nanofiber matrices are popularly used for a variety of tissue engineering applications.

Amyloid fibrils are self-assembling proteins that can be used as biomaterials due to their molecular and hierarchical structure, making them resilient to mechanical conditioning. Similar to amyloid fibrils, naturally derived proteins and short peptides can be fabricated into thin fibers which can be used for various biomedical applications. Techniques such as self-assembly, wet-spinning, and electrospinning can be used to fabricate protein fibers. Even though self-assembly and wet-spinning have been used to successfully produce protein fibers, these techniques provide limited control over fiber morphology. Electrospinning is a versatile method for spinning synthetic and natural polymers. It produces fibrous matrices with high porosity, interconnectivity, and volume to mass ratio. This chapter will summarize the basic principle behind electrospinning, parameters that can be optimized to produce fibrous matrices with specific morphology, and examples of proteins such as silk fibroin, collagen, and albumin that have been electrospun for applications such as tissue engineering, wound healing, drug delivery, and biosensors.

Allografts are commonly used as an alternative to autografts for bone defect repair, but in some instances suffer from reduced host incorporation that can lead to complications 5 and 10 years post-implantation. We have previously demonstrated that using a thin polymeric coating along the outer surfaces of cortical bone can improve the biofunctionality of devitalized bone allografts by permitting the upload and delivery of growth factors from the thin coating. Here, we expand on our previous work to develop a methodology to coat porous trabecular allografts with a similar polymer coating with careful attention to maintaining an open pore network within the allograft and establishing the effect of varying the polymer solution viscosity on coating extent and thickness. Results indicate that varying the polymer solution viscosity by changing the polymer to solvent ratio used to coat the samples allows for variations in coating thickness and extent of surface area coverage, each perhaps allowing for greater control over growth factor loading and release concentration and kinetics. Further, we develop a dynamic coating method that produces a continuous and consistent coating throughout the pore structure vs. the previously used static method that resulted in a discontinuous coating throughout the pore network. The work demonstrated here provides important techniques for varying the payload of polymer-coated, factor-loaded trabecular allografts which would provide another tool to the orthopedic surgeon when healing large-scale defects with trabecular allograft. Large scale bone defects are traditionally treated with either autografts or allografts. Allografts suffer from poor incorporation in part due to the processing undertaken to render them safe to transplant from one individual to another. We sought to add back some of the biological functionality that is removed in this processing by loading growth factors into a very thin polymeric coating that we apply to the surface of the allograft. Our results indicate a thin, continuous polymeric coating throughout the trabecular allograft, with the amount of this coating varying as we vary the concentration of this polymer solution prior to coating. Future studies will evaluate the ability of this varied concentration of polymer coating to permit the delivery of varying amounts of growth factors, imparting a greater degree of control over the amount of growth factor delivered from the coated allografts.

A biodegradable scaffold with tissue ingrowth and load-bearing capabilities is required to accelerate the healing of bone defects. However, it is difficult to maintain the mechanical properties as well as biodegradability and porosity (necessary for bone ingrowth) at the same time. Therefore, in the present study, polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA5050) were mixed in varying ratio and incorporated with 20 wt.% βTCP. The mixture was shaped under pressure into originally non-porous cylindrical constructs. It is envisioned that the fabricated constructs will develop porosity with the time-dependent biodegradation of the polymer blend. The mechanical properties will be sustained since the decrease in mechanical properties associated with the dissolution of the PLGA and the formation of the porous structure will be compensated with the new bone formation and ingrowth. To prove the hypothesis, we have systematically studied the effects of samples composition on the time-dependent dissolution behavior, pore formation, and mechanical properties of the engineered samples, in vitro. The highest initial (of as-prepared samples) values of the yield strength (0.021±0.002 GPa) and the Young's modulus (0.829±0.096 GPa) were exhibited by the samples containing 75 wt.% of PLGA. Increase of the PLGA concentration from 25 wt.% to 75 wt.% increased the rate of biodegradation by a factor of 3 upon 2 weeks in phosphate buffered saline (1× PBS). The overall porosity and the pore sizes increased with the dissolution time indicating that the formation of in-situ pores can indeed enable the migration of cells followed by vascularization and bone growth.

Summary Background Accelerating orthodontic tooth movement (OTM) through biologically effective methods, such as increasing osteoclast-mediated alveolar resorption, could effectively shorten treatment time. Objective To evaluate an injectable formulation containing receptor activator of nuclear factor kappa-B ligand (RANKL) on the OTM. Materials and methods We fabricated a RANKL formulation from 100 µl of 100 µg/ml RANKL adsorbed on 10 mg of poly(lactic acid-co-glycolic acid) microspheres embedded in a 10 wt% aqueous hydroxyethyl cellulose carrier gel. We characterized these formulations for the rate of RANKL release, and then tested for bioactivity using in vitro cell culture. In vivo OTM studies were conducted using 15 week old male Wistar rats for 14 days. We injected the RANKL formulations palatal to the left maxillary first molar and accomplished OTM with a nickel–titanium (NiTi) coil spring applying 5–8 g force. Control groups involved the application of NiTi coil spring with and without placebo formulation. The outcome measure included the distance of tooth movement, bone volume fraction, tissue density, and root volume determined with micro-computed tomography. We determined the amount of osteoclast activity using tartrate-resistant acid phosphatase (TRAP) staining. Results These formulations were able to sustain the release of RANKL for more than 30 days, and the released RANKL showed a positive effect on mice osteoclast precursor cells (RAW 264.7). Reported injectable RANKL formulations were effective in accelerating OTM compared with other control groups, with 129.2 per cent more tooth movement than no formulation and 71.8 per cent more than placebo formulation, corresponding with a significant increase in the amount of TRAP activity. We did not observe any significant differences in root resorption between the groups. Conclusion Our study shows a significant increase in OTM with injectable formulations containing RANKL.

This article reviews the current status of skin tissue equivalents that have emerged as relevant tools in commercial and therapeutic product development applications. Due to rise of animal welfare concerns, numerous companies have designed skin model alternatives to assess the efficacy of pharmaceutical, skincare and cosmetic products in an in vitro setting, decreasing the dependency on such methods. Several validated test methods are performed on such models to investigate various characteristics including skin irritation, permeation, corrosion and hydration, genotoxicity and absorption. Skin models have also made an impact in determining the root causes of skin diseases and providing a means for developing an effective treatment in certain clinical applications. When designing a skin model there are various chemical and physical considerations that need to be considered to produce a biomimetic design. This includes designing a structure that mimics the structural characteristics and mechanical strength needed for tribological property measurement and toxicological testing. Presently, various commercial products have made significant progress towards achieving a native skin alternative, with focuses on one or more of the aforementioned considerations. Further research perspectives involve development of a functional bi-layered model that mimics the constituent properties such as viscoelasticity and organization of the native epidermis and dermis, as well as incorporation of dynamic elements to mimic skin’s interactions with other organs. In this article, the skin models are divided into three categories: In Vitro Epidermal Skin Equivalents, In Vitro Full Thickness Skin Equivalents, and Clinical Skin Equivalents. A description of skin model characteristics, testing methods, applications, and potential improvements is presented.

Use of growth factors as biochemical molecules to elicit cellular differentiation is a common strategy in tissue engineering. However, limitations associated with growth factors, such as short half-life, high effective physiological doses, and high costs, have prompted the search for growth factor alternatives, such as growth factor mimics and other proteins. This work explores the use of insulin protein as a biochemical factor to aid in tendon healing and differentiation of cells on a biomimetic electrospun micro-nanostructured scaffold. Dose response studies were conducted using human mesenchymal stem cells (MSCs) in basal media supplemented with varied insulin concentrations. A dose of 100-ng/mL insulin showed increased expression of tendon markers. Synthetic-natural blends of various ratios of polycaprolactone (PCL) and cellulose acetate (CA) were used to fabricate micro-nanofibers to balance physicochemical properties of the scaffolds in terms of mechanical strength, hydrophilicity, and insulin delivery. A 75:25 ratio of PCL:CA was found to be optimal in promoting cellular attachment and insulin immobilization. Insulin insulin deliveryimmobilized fiber matrices also showed increased expression of tendon phenotypic markers by MSCs similar to findings with insulin supplemented media, indicating preservation of insulin bioactivity. Insulin functionalized scaffolds may have potential applications in tendon healing and regeneration.

Peripheral nerve injury accounts for roughly 2.8% of all trauma patients with an annual cost of 7 billion USD in the U.S. alone. Current treatment options rely on surgical intervention with the use of an autograft, despite associated shortcomings. Engineered nerve guidance conduits, stem cell therapies, and transient electrical stimulation have reported to increase speeds of functional recovery. As an alternative to the conduction effects of electrical stimulation, we have designed and optimized a nerve guidance conduit with aligned microchannels for the sustained release of a small molecule drug that promotes nerve impulse conduction. A biodegradable chitosan structure reinforced with drug-loaded halloysite nanotubes (HNT) was formed into a foam-like conduit with interconnected, longitudinally-aligned pores with an average pore size of 59.3 ± 14.2 μm. The aligned composite with HNTs produced anisotropic mechanical behavior with a Young's modulus of 0.33 ± 0.1 MPa, very similar to that of native peripheral nerve. This manuscript reports on the sustained delivery of 4-Aminopyridine (4AP, molecular weight 94.1146 g/mol), a potassium-channel blocker as a growth factor alternative to enhance the rate of nerve regeneration. The conduit formulation released a total of 30 ± 2% of the encapsulated 4AP in the first 7 days. Human Schwann cells showed elevated expression of key proteins such as nerve growth factor, myelin protein zero, and brain derived neurotrophic factor in a 4AP dose dependent manner. Preliminary in vivo studies in a critical-sized sciatic nerve defect in Wistar rats confirmed conduit suturability and strength to withstand ambulatory forces over 4 weeks of their implantation. Histological evaluations suggest conduit biocompatibility and Schwann cell infiltration and organization within the conduit and lumen. These nerve guidance conduits and 4AP sustained delivery may serve as an attractive strategy for nerve repair and regeneration.

Tissue engineering is a field of study that focuses on the integration of scaffolds, cells, and bioactive factors to produce functional biological tissues. Novel biomaterials provide the foundation for the development of tissue engineering and regenerative medicine. Through the use and improvement of these biocompatible materials, biomedical engineers and researchers have been able to develop treatments that address unmet clinical needs as well as improve earlier practices. These treatments address a wide range of disciplines from musculoskeletal applications to tissue regeneration. An advantage is that the Food and Drug Administration has approved several biomaterials, both natural and synthetic, as compliant for use in the human body. This prior approval enables researchers to improve the use of biomaterials in their approved applications, and advance the quality of treatment in a shorter period of time. Scientists, engineers, and clinicians all work in a collaborative effort to design treatments with biomaterials that closely resemble human tissues, and promote optimal repair or regeneration at the targeted site. This article will describe some of the most commonly used natural and synthetic biomaterials including their uses, advantages, disadvantages, and applications for various tissue engineering and regenerative medicine applications.

Successful tissue regeneration strategies focus on the use of novel biomaterials, structures, and a variety of cues to control cell behavior and promote regeneration. Studies discovered how biomaterial/ structure cues in the form of biomaterial chemistry, material stiffness, surface topography, pore, and degradation properties play an important role in controlling cellular events in the contest of in vitro and in vivo tissue regeneration. Advanced biomaterials structures and strategies are developed to focus on the delivery of bioactive factors, such as proteins, peptides, and even small molecules to influence cell behavior and regeneration. The present article is an effort to summarize important findings and further discuss biomaterial strategies to influence and control cell behavior directly via physical and chemical cues. This article also touches on various modern methods in biomaterials processing to include bioactive factors as signaling cues to program cell behavior for tissue engineering and regenerative medicine.

A variety of engineered scaffolds have been created for tissue engineering using polymers, ceramics and their composites. Biomimicry has been adopted for majority of the three-dimensional (3D) scaffold design both in terms of physicochemical properties, as well as bioactivity for superior tissue regeneration. Scaffolds fabricated via salt leaching, particle sintering, hydrogels and lithography have been successful in promoting cell growth in vitro and tissue regeneration in vivo. Scaffold systems derived from decellularization of whole organs or tissues has been popular due to their assured biocompatibility and bioactivity. Traditional scaffold fabrication techniques often failed to create intricate structures with greater resolution, not reproducible and involved multiple steps. The 3D printing technology overcome several limitations of the traditional techniques and made it easier to adopt several thermoplastics and hydrogels to create micro-nanostructured scaffolds and devices for tissue engineering and drug delivery. This review highlights scaffold fabrication methodologies with a focus on optimizing scaffold performance through the matrix pores, bioactivity and degradation rate to enable tissue regeneration. Review highlights few examples of bioactive scaffold mediated nerve, muscle, tendon/ligament and bone regeneration. Regardless of the efforts required for optimization, a shift in 3D scaffold uses from the laboratory into everyday life is expected in the near future as some of the methods discussed in this review become more streamlined., Graphical abstract Image 1, Highlights • The requirements for an ideal biomaterial-based scaffold are highlighted. • The relationship between mechanical properties and porosity is discussed. • Several scaffold fabrication techniques are discussed with advantages and disadvantages. • Examples of tissue engineering strategies provided

Therapeutic biomolecules often require frequent administration and supramolecular dosing to achieve therapeutic efficiencies and direct infusion into treatment or defect sites results in inadequate physiological response and at times severe side effects or mis-targeting. Delivery systems serve several purposes such as increased circulatory time, increased biomolecule half-life, and incorporation of new innovations can enable highly specific cell targeting and improved cell and nucleus permeability. Poly(lactic acid) (PLA) has become a "material of choice" due to wide availability, reproducible synthetic route, customization, versatility, biodegradability and biocompatibility. Furthermore, PLA is amenable to a variety of fabrication methodologies and chemistries allowing an expansive library correlating physio-chemical properties, characteristics, and applications. This article discusses challenges to biomolecule delivery, and classical approaches of PLA based biomolecule delivery and targeting strategies under development and in trials.

This manuscript reports the characterization of molecularly self-assembled collagen nanofibers on a natural polymeric microporous structure and their ability to support stem cell differentiation in vitro and host tissue response in vivo. Specifically, cellulose acetate (CA) and poly(lactic acid-co-glycolic acid) (PLGA) produced two different microporous structures that were coated with self-assembled type I collagen (PLGAc and CAc). Though the total content of collagen was similar between the two materials after coating, the material chemistries significantly affected the molecular collagen self-assembly resulting in a more biomimetic nanofibrillar D-banding pattern on CA (mean fiber diameter of 80 nm) and a sheet like coating on PLGA (mean fiber diameter of 150 nm). Human mesenchymal stem cells (hMSCs) cultured on CA and CAc showed a significantly higher degree of in vitro osteoblastic progression, in contrast to PLGA and PLGAc. Furthermore, at 2 weeks post subcutaneous implantation, collagen coated CA materials showed increased matrix cellularization and enhanced biocompatibility. At 12 weeks both CA and CAc showed significantly greater matrix cellularity and immune acceptance compared to PLGA. This work illustrates the role of materials chemistry to dictate nanoscale protein assembly and its effect in terms of in vitro stem cell differentiation and in vivo host immune response. We also have proved that inclusion of nanoscale self-assembled ECM components such as collagen can enhance the stem cell inductive capabilities and biocompatibility of hydrophilic natural polymeric materials, making them viable alternatives to widely used synthetic polymers.

Engineered soft tissue products-both tendon and ligament-have gained tremendous interest in regenerative medicine as alternatives to autograft and allograft treatments due to their potential to overcome limitations such as pain and donor site morbidity. Tendon engineered grafts have focused on the replication of native tendon tissue composition and architecture in the form of scaffolds using synthetic or natural biomaterials seeded with cells and factors. However, these approaches suffer due to static culture environments that fail to mimic the dynamic tissue environment and mechanical forces required to promote tenogenic differentiation of cultured cells. Mechanical stimulation is sensed by cellular mechanosensors such as integrins, focal adhesion kinase, and other transmembrane receptors which promote tenogenic gene expression and synthesis of tendon extracellular matrix components such as Type I collagen. Thus, it is imperative to apply biological and biomechanical aspects to engineer tendon. This review highlights the origin of tendon tissue, its ability to sense forces from its microenvironment, and the biological machinery that helps in mechanosensation. Additionally, this review focuses on use of bioreactors that aid in understanding cell-microenvironment interactions and enable the design of mechanically competent tendon tissue. We categorize these bioreactors based on functional features, sample size/type, and loading regimes and discuss their application in tendon research. The objective of this article is to provide a perspective on biomechanical considerations in the development of functional tendon tissue.

Anterior cruciate ligament (ACL) plays a crucial role stabilizing the knee joint while connecting tibia to femur. Lack of proper treatment of injured ACL can lead to meniscus tear and osteoarthritis. Interference screws secure the graft tissue for superior integration of graft on host tissue during autograft fixation. Metal interference screws come with various disadvantages like mechanical load mismatch, graft laceration, secondary surgical removal and hindrance during MRI and CT post-operative scan. Though biodegradable polymeric screws provide various advantages their clinical outcomes reveal unprecedented complications for long term use of such screws. This review highlights polymer and composite screw currently available for surgical fixations and associated adverse reactions with the proposed mechanism for tunnel enlargement, effusion, osteolysis in ligament repairs. The need for suitable material engineering for development of orthopedic screws for successful rigid fixation has been highlighted in this review.