Structural Biology of the Tumor Microenvironment.

Cancers can be described as "rogue organs" (Balkwill FR, Capasso M, Hagemann T, J Cell Sci 125:5591-5596, 2012) because they are composed of multiple cell types and tissues. The transformed cells can recruit and alter healthy cells from surrounding tissues for their own benefit. It is these interactions that create the tumor microenvironment (TME). The TME describes the cells, factors, and extracellular matrix proteins that make up the tumor and the area around it; the biology of the TME influences tumor progression. Changes in the TME can lead to the growth and development of the tumor, the death of the tumor, or tumor metastasis. Metastasis is the process by which cancer spreads from its initial site to a different part of the body. Metastasis occurs when cancer cells enter the circulatory system or lymphatic system after they break away from a tumor. Once the cells leave, they can travel to a different part of the body and form new tumors. Therefore, understanding the TME is critical to fully understand cancer and find a way to successfully combat it. Knowledge of the TME can better inform researchers of the ability of potential therapies to reach tumor cells. It can also give researchers potential targets to kill the tumor. Instead of directly killing the cancer cells, therapies can target an aspect of the TME which could then halt tumor development or lead to tumor death. In other cases, targeting another aspect of the TME could make it easier for another therapy to kill the cancer cells, for example, using nanoparticles with collagenases to target the collagen in the surrounding environment to expose the cancer cells to drugs (Zinger A, et al, ACS Nano 13(10):11008-11021, 2019).The TME can be split simply into cells and the structural matrix. Within these groups are fibroblasts, structural proteins, immune cells, lymphocytes, bone marrow-derived inflammatory cells, blood vessels, and signaling molecules (Spill F, et al, Curr Opin Biotechnol 40:41-48, 2016; Del Prete A, et al, Curr Opin Pharmacol 35:40-47, 2017; Arneth B, Medicina (Kaunas) 56(1), 2019). From structure to providing nutrients for growth, each of these components plays a critical role in tumor maintenance. Together these components impact cancer growth, development, and resistance to therapies (Hanahan D, Coussens LM, Cancer Cell 21:309-322, 2012). In this chapter, we will describe the TME and express the importance of the cellular and structural elements of the TME.

Characterization and optimization of actuating poly(ethylene glycol) diacrylate/acrylic acid hydrogels as artificial muscles.

Large volume deficiencies in skeletal muscle tissue fail to heal with conservative treatments, and improved treatment methods are needed. Tissue engineered scaffolds for skeletal muscle need to mimic the optimal environment for muscle development by providing the proper electric, mechanical, and chemical cues. Electroactive polymers, polymers that change in size or shape in response to an electric field, may be able to provide the optimal environment for muscle growth. In this study, an electroactive polymer made from poly(ethylene glycol) diacrylate (PEGDA) and acrylic acid (AA) is characterized and optimized for movement and biocompatibility. Hydrogel sample thickness, overall polymer concentration, and the ratio of PEGDA to AA were found to significantly impact the actuation response. C2C12 mouse myoblast cells attached and proliferated on hydrogel samples with various ratios of PEGDA to AA. Future experiments will produce hydrogel samples combined with aligned guidance cues in the form of electrospun fibers to provide a favorable environment for muscle development.

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro.

BACKGROUND: Proliferative therapy, or prolotherapy, is a controversial treatment method for many connective tissue injuries and disorders. It involves the injection of a proliferant, or irritant solution, into the site of injury, which causes small-scale cell death. This therapeutic trauma is theorized to initiate the body's wound-healing cascade, perhaps leading to tissue repair. The immediate effects of many of these proliferants are poorly characterized, as are the cellular responses to them; here, we sought to evaluate the immediate effects of two common proliferants (dextrose and P2G, a combination of phenol, glucose, and glycerin) on the cellular response of human tenocytes, and begin to explicate the mechanisms with which each proliferant functions. QUESTIONS/PURPOSES: We asked: What are the effects of treating cultured tenocytes with proliferative treatment agents on their (1) cellular metabolic activity, (2) RNA expression, (3) protein secretion, and (4) cell migration? METHODS: Using human hamstring and Achilles tendon cells, we attempted to answer our research questions. We used a colorimetric metabolic assay to assess the effect of dextrose and P2G proliferant treatment on cell mitochondrial activity compared with nontreated tenocytes. Next, using quantitative PCR, ELISA, and a reporter cell line, we assessed the expression of several key markers involved in tendon development and inflammation. In addition, we used a scratch wound-healing assay to evaluate the effect of proliferant treatment on tenocyte migration. RESULTS: Results showed that exposure to both solutions led to decreased metabolic activity of tenocytes, with P2G having the more pronounced effect (75% ± 7% versus 95% ± 7% of untreated control cell metabolic levels) (ANOVA; p < 0.01; mean difference, 0.202; 95% CI, 0.052-0.35). Next, gene expression analysis confirmed that treatment led to the upregulation of key proinflammatory markers including interleukin-8 and cyclooxygenase-2 and downregulation of the matrix marker collagen type I. Furthermore, using a reporter cell line for transforming growth factor-ß (TGF-ß), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-ß bioactivity. Analysis of soluble proteins using ELISA revealed elevated levels of soluble prostaglandin E2 (PGE2), a prominent inducer of inflammation. Finally, both solutions led to decreased cellular migration in the tenocytes. CONCLUSIONS: Taken together, these results suggest that prolotherapy, more so with P2G, may work by decreasing cellular function and eliciting an inflammatory response in tenocytes. Additional studies are needed to confirm the cellular signaling mechanisms involved and the resulting immediate response in vivo. CLINICAL RELEVANCE: If these preliminary in vitro findings can be confirmed in an in vivo model, they may provide clues for a possible cellular mechanism of a common alternative treatment method currently used for certain soft tissue injuries.

Cross-Talk Between Human Tenocytes and Bone Marrow Stromal Cells Potentiates Extracellular Matrix Remodeling In Vitro.

Tendon and ligament (T/L) pathologies account for a significant portion of musculoskeletal injuries and disorders. Tissue engineering has emerged as a promising solution in the regeneration of both tissues. Specifically, the use of multipotent human mesenchymal stromal cells (hMSC) has shown great promise to serve as both a suitable cell source for tenogenic regeneration and a source of trophic factors to induce tenogenesis. Using four donor sets, we investigated the bidirectional paracrine tenogenic response between human hamstring tenocytes (hHT) and bone marrow-derived hMSC. Cell metabolic assays showed that only one hHT donor experienced sustained notable increases in cell metabolic activity during co-culture. Histological staining confirmed that co-culture induced elevated collagen protein levels in both cell types at varying time-points in two of four donor sets assessed. Gene expression analysis using qPCR showed the varied up-regulation of anabolic and catabolic markers involved in extracellular matrix maintenance for hMSC and hHT. Furthermore, analysis of hMSC/hHT co-culture secretome using a reporter cell line for TGF-ß, a potent inducer of tenogenesis, revealed a trend of higher TGF-ß bioactivity in hMSC secretome compared to hHT. Finally, hHT cytoskeletal immunostaining confirmed that both cell types released soluble factors capable of inducing favorable tenogenic morphology, comparable to control levels of soluble TGF-ß1. These results suggest a potential for TGF-ß-mediated signaling mechanism that is involved during the paracrine interplay between the two cell types that is reminiscent of T/L matrix remodeling/turnover. These findings have significant implications in the clinical use of hMSC for common T/L pathologies.

An overview of recent patents on musculoskeletal interface tissue engineering.

Interface tissue engineering involves the development of engineered grafts that promote integration between multiple tissue types. Musculoskeletal tissue interfaces are critical to the safe and efficient transmission of mechanical forces between multiple musculoskeletal tissues, e.g., between ligament and bone tissue. However, these interfaces often do not physiologically regenerate upon injury, resulting in impaired tissue function. Therefore, interface tissue engineering approaches are considered to be particularly relevant for the structural restoration of musculoskeletal tissues interfaces. In this article, we provide an overview of the various strategies used for engineering musculoskeletal tissue interfaces with a specific focus on the recent important patents that have been issued for inventions that were specifically designed for engineering musculoskeletal interfaces as well as those that show promise to be adapted for this purpose.

Quantification of strain induced damage in medial collateral ligaments.

In the past years, there have been several experimental studies that aimed at quantifying the material properties of articular ligaments such as tangent modulus, tensile strength, and ultimate strain. Little has been done to describe their response to mechanical stimuli that lead to damage. The purpose of this experimental study was to characterize strain-induced damage in medial collateral ligaments (MCLs). Displacement-controlled tensile tests were performed on 30 MCLs harvested from Sprague Dawley rats. Each ligament was monotonically pulled to several increasing levels of displacement until complete failure occurred. The stress-strain data collected from the mechanical tests were analyzed to determine the onset of damage and its evolution. Unrecoverable changes such as increase in ligament's elongation at preload and decrease in the tangent modulus of the linear region of the stress-strain curves indicated the occurrence of damage. Interestingly, these changes were found to appear at two significantly different threshold strains (P<0.05). The mean threshold strain that determined the increase in ligament's elongation at preload was found to be 2.84% (standard deviation (SD) = 1.29%) and the mean threshold strain that caused the decrease in the tangent modulus of the linear region was computed to be 5.51% (SD = 2.10%), respectively. The findings of this study suggest that the damage mechanisms associated with the increase in ligament's elongation at preload and decrease in the tangent modulus of the linear region in the stress-strain curves in MCLs are likely different.

Tissue Engineered 3D Constructs for Volumetric Muscle Loss.

Severe injuries to skeletal muscles, including cases of volumetric muscle loss (VML), are linked to substantial tissue damage, resulting in functional impairment and lasting disability. While skeletal muscle can regenerate following minor damage, extensive tissue loss in VML disrupts the natural regenerative capacity of the affected muscle tissue. Existing clinical approaches for VML, such as soft-tissue reconstruction and advanced bracing methods, need to be revised to restore tissue function and are associated with limitations in tissue availability and donor-site complications. Advancements in tissue engineering (TE), particularly in scaffold design and the delivery of cells and growth factors, show promising potential for regenerating damaged skeletal muscle tissue and restoring function. This article provides a brief overview of the pathophysiology of VML and critiques the shortcomings of current treatments. The subsequent section focuses on the criteria for designing TE scaffolds, offering insights into various natural and synthetic biomaterials and cell types for effectively regenerating skeletal muscle. We also review multiple TE strategies involving both acellular and cellular scaffolds to encourage the development and maturation of muscle tissue and facilitate integration, vascularization, and innervation. Finally, the article explores technical challenges hindering successful translation into clinical applications.

Rational design of poly(peptide-ester) block copolymers for enzyme-specific surface resorption.

Tissue resorption and remodeling are pivotal steps in successful healing and regeneration, and it is important to design biomaterials that are responsive to regenerative processes in native tissue. The cell types responsible for remodeling, such as macrophages in the soft tissue wound environment and osteoclasts in the bone environment, utilize a class of enzymes called proteases to degrade the organic matrix. Many hydrophobic thermoplastics used in tissue regeneration are designed to degrade and resorb passively through hydrolytic mechanisms, leaving the potential of proteolytic-guided degradation underutilized. Here, we report the design and synthesis of a tyrosol-derived peptide-polyester block copolymer where protease-mediated resorption is tuned through changing the chemistry of the base polymer backbone and protease specificity is imparted through incorporation of specific peptide sequences. Quartz crystal microbalance was used to quantify polymer surface resorption upon exposure to various enzymes. Aqueous solubility of the diacids and the thermal properties of the resulting polymer had a significant effect on enzyme-mediated polymer resorption. While peptide incorporation at 2 mol% had little effect on the final thermal and physical properties of the block copolymers, its incorporation improved polymer resorption significantly in a peptide sequence- and protease-specific manner. To our knowledge, this is the first example of a peptide-incorporated linear thermoplastic with protease-specific sensitivity reported in the literature. The product is a modular system for engineering specificity in how polyesters can resorb under physiological conditions, thus providing a potential framework for improving vascularization and integration of biomaterials used in tissue engineering.

The Use of Collagen Methacrylate in Actuating Polyethylene Glycol Diacrylate-Acrylic Acid Scaffolds for Muscle Regeneration.

After muscle loss or injury, skeletal muscle tissue has the ability to regenerate and return its function. However, large volume defects in skeletal muscle tissue pose a challenge to regenerate due to the absence of regenerative elements such as biophysical and biochemical cues, making the development of new treatments necessary. One potential solution is to utilize electroactive polymers that can change size or shape in response to an external electric field. Poly(ethylene glycol) diacrylate (PEGDA) is one such polymer, which holds great potential as a scaffold for muscle tissue regeneration due to its mechanical properties. In addition, the versatile chemistry of this polymer allows for the conjugation of new functional groups to enhance its electroactive properties and biocompatibility. Herein, we have developed an electroactive copolymer of PEGDA and acrylic acid (AA) in combination with collagen methacrylate (CMA) to promote cell adhesion and proliferation. The electroactive properties of the CMA + PEGDA:AA constructs were investigated through actuation studies. Furthermore, the biological properties of the hydrogel were investigated in a 14-day in vitro study to evaluate myosin light chain (MLC) expression and metabolic activity of C2C12 mouse myoblast cells. The addition of CMA improved some aspects of material bioactivity, such as MLC expression in C2C12 mouse myoblast cells. However, the incorporation of CMA in the PEGDA:AA hydrogels reduced the sample movement when placed under an electric field, possibly due to steric hindrance from the CMA. Further research is needed to optimize the use of CMA in combination with PEGDA:AA as a potential scaffold for skeletal muscle tissue engineering.

Cross-talk between endothelial and breast cancer cells regulates reciprocal expression of angiogenic factors in vitro.

Reciprocal growth factor exchange between endothelial and malignant cells within the tumor microenvironment may directly stimulate neovascularization; however, the role of host vasculature in regulating tumor cell activity is not well understood. While previous studies have examined the angiogenic response of endothelial cells to tumor-secreted factors, few have explored tumor response to endothelial cells. Using an in vitro co-culture system, we investigated the influence of endothelial cells on the angiogenic phenotype of breast cancer cells. Specifically, VEGF, ANG1, and ANG2 gene and protein expression were assessed. When co-cultured with microvascular endothelial cells (HMEC-1), breast cancer cells (MDA-MB-231) significantly increased expression of ANG2 mRNA (20-fold relative to MDA-MB-231 monoculture). Moreover, MDA-MB-231/HMEC-1 co-cultures produced significantly increased levels of ANG2 (up to 580 pg/ml) and VEGF protein (up to 38,400 pg/ml) while ANG1 protein expression was decreased relative to MDA-MB-231 monocultures. Thus, the ratio of ANG1:ANG2 protein, a critical indicator of neovascularization, shifted in favor of ANG2, a phenomenon known to correlate with vessel destabilization and sprouting in vivo. This angiogenic response was not observed in nonmalignant breast epithelial cells (MCF-10A), where absolute protein levels of MCF-10A/HMEC-1 co-cultures were an order of magnitude less than that of the MDA-MB-231/HMEC-1 co-cultures. Results were further verified with a functional angiogenesis assay demonstrating well-defined microvascular endothelial cell (TIME) tube formation when cultured in media collected from MDA-MB-231/HMEC-1 co-cultures. This study demonstrates that the angiogenic activity of malignant mammary epithelial cells is significantly enhanced by the presence of endothelial cells.

Elastic and viscoelastic properties of a type I collagen fiber.

A new mathematical model is presented to describe the elastic and viscoelastic properties of a single collagen fiber. The model is formulated by accounting for the mechanical contribution of the collagen fiber's main constituents: the microfibrils, the interfibrillar matrix and crosslinks. The collagen fiber is modeled as a linear elastic spring, which represents the mechanical contribution of the microfibrils, and an arrangement in parallel of elastic springs and viscous dashpots, which represent the mechanical contributions of the crosslinks and interfibrillar matrix, respectively. The linear elastic spring and the arrangement in parallel of elastic springs and viscous dashpots are then connected in series. The crosslinks are assumed to gradually break under strain and, consequently, the interfibrillar is assumed to change its viscous properties. Incremental stress relaxation tests are conducted on dry collagen fibers reconstituted from rat tail tendons to determine their elastic and viscoelastic properties. The elastic and total stress-strain curves and the stress relaxation at different levels of strain collected by performing these tests are then used to estimate the parameters of the model and evaluate its predictive capabilities.

Design and analysis of braid-twist collagen scaffolds.

Collagen type I fiber-based scaffolds for anterior cruciate ligament (ACL) replacement were evaluated for their mechanical properties and their ability to promote cellular proliferation. Prior to scaffold formation, two crosslinking methods were investigated on individual reconstituted collagen type I fibers, ultraviolet radiation, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Crosslinking with EDC for 4 hr yielded mechanical properties similar to the human ACL; therefore, scaffold crosslinking was done with EDC for 4 hr. A braid-twist scaffold design was used, and scaffolds were left uncrosslinked, crosslinked after the addition of gelatin, or crosslinked without gelatin. The ultimate tensile strength, Young's modulus, and viscoelastic properties of the scaffolds were then evaluated. In order to assess cellular response on the scaffolds, primary rat ligament fibroblast cells were seeded upon the scaffolds. Cell activity was evaluated at days 7, 14, and 21 using a Cell Titer 96(®) AQueous One Solution Cell Proliferation Assay (MTS Assay). The mechanical testing results showed that among the three scaffold groups, the crosslinked scaffolds without gelatin displayed an ultimate tensile strength, Young's modulus, and viscoelastic properties that were closest to the human ACL. Improvements are still desired to enhance the mechanical compliance and ductility of these scaffolds. Cell activity was observed on all cell-seeded scaffolds by day 7, but by day 21 only the crosslinked scaffolds without gelatin displayed increased cellular activity compared with the negative controls. Although improvement is still needed, the results suggest that these scaffolds have the potential to contribute toward an ACL replacement strategy.

Intra-articular injection of epigallocatechin (EGCG) crosslinks and alters biomechanical properties of articular cartilage, a study via nanoindentation.

Osteoarthritis and rheumatoid arthritis are debilitating conditions, affecting millions of people. Both osteoarthritis and rheumatoid arthritis degrade the articular cartilage (AC) at the ends of long bones, resulting in weakened tissue prone to further damage. This degradation impairs the cartilage's mechanical properties leading to areas of thinned cartilage and exposed bone which compromises the integrity of the joint. No preventative measures exist for joint destruction. Discovering a way to slow the degradation of AC or prevent it would slow the painful progression of the disease, allowing millions to live pain-free. Recently, that the articular injection of the polyphenol epigallocatechin-gallate (EGCG) slows AC damage in an arthritis rat model. It was suggested that EGCG crosslinks AC and makes it resistant to degradation. However, direct evidence that intraarticular injection of EGCG crosslinks cartilage collagen and changes its compressive properties are not known. The aim of this study was to investigate the effects of intraarticular injection of EGCG induced biomechanical properties of AC. We hypothesize that in vivo exposure EGCG will bind and crosslink to AC collagen and alter its biomechanical properties. We developed a technique of nano-indentation to investigate articular cartilage properties by measuring cartilage compressive properties and quantifying differences due to EGCG exposure. In this study, the rat knee joint was subjected to a series of intraarticular injections of EGCG and contralateral knee joint was injected with saline. After the injections animals were sacrificed, and the knees were removed and tested in an anatomically relevant model of nanoindentation. All mechanical data was normalized to the measurements in the contralateral knee to better compare data between the animals. The data demonstrated significant increases for reduced elastic modulus (57.5%), hardness (83.2%), and stiffness (17.6%) in cartilage treated with injections of EGCG normalized to those treated with just saline solution when compared to baseline subjects without injections, with a significance level of alpha = 0.05. This data provides evidence that EGCG treated cartilage yields a strengthened cartilage matrix as compared to AC from the saline injected knees. These findings are significant because the increase in cartilage biomechanics will translate into resistance to degradation in arthritis. Furthermore, the data suggest for the first time that it is possible to strengthen the articular cartilage by intraarticular injections of polyphenols. Although this data is preliminary, it suggests that clinical applications of EGCG treated cartilage could yield strengthened tissue with the potential to resist or compensate for matrix degradation caused by arthritis.

Uric acid released from poly(ε-caprolactone) fibers as a treatment platform for spinal cord injury.

Spinal cord injury (SCI) is characterized by a primary mechanical phase of injury, resulting in physical tissue damage, and a secondary pathological phase, characterized by biochemical processes contributing to inflammation, neuronal death, and axonal demyelination. Glutamate-induced excitotoxicity (GIE), in which excess glutamate is released into synapses and overstimulates glutamate receptors, is a major event in secondary SCI. GIE leads to mitochondrial damage and dysfunction, release of reactive oxygen species (ROS), DNA damage, and cell death. There is no clinical treatment that targets GIE after SCI, and there is a need for therapeutic targets for secondary damage in patients. Uric acid (UA) acts as an antioxidant and scavenges free radicals, upregulates glutamate transporters on astrocytes, and preserves neuronal viability in in vitro and in vivo SCI models, making it a promising therapeutic candidate. However, development of a drug release platform that delivers UA locally to the injured region in a controlled manner is crucial, as high systemic UA concentrations can be detrimental. Here, we used the electrospinning technique to synthesize UA-containing poly(ɛ-caprolactone) fiber mats that are biodegradable, biocompatible, and have a tunable degradation rate. We optimized delivery of UA as a burst within 20 min from uncoated fibers and sustained release over 2 h with poly(ethylene glycol) diacrylate coating. We found that both of these fibers protected neurons and decreased ROS generation from GIE in organotypic spinal cord slice culture. Thus, fiber mats represent a promising therapeutic for UA release to treat patients who have suffered a SCI.

Elastic energy storage in an unmineralized collagen type I molecular model with explicit solvation and water infiltration.

Collagen type I is a structural protein that provides tensile strength to tendons and ligaments. Type I collagen molecules form collagen fibers, which are viscoelastic and can therefore store energy elastically via molecular elongation and dissipate viscous energy through molecular rearrangement and fibrillar slippage. The ability to store elastic energy is important for the resiliency of tendons and ligaments, which must be able to deform and revert to their initial lengths with changes in load. In an earlier paper by one of the present authors, molecular modeling was used to investigate the role of mineralization upon elastic energy storage in collagen type I. Their collagen model showed a similar trend to their experimental data but with an over-estimation of elastic energy storage. Their simulations were conducted in vacuum and employed a distance-dependent dielectric function. In this study, we performed a re-evaluation of Freeman and Silver's model data incorporating the effects of explicit solvation and water infiltration, in order to determine whether the model data could be improved with a more accurate representation of the solvent and osmotic effects. We observed an average decrease in the model's elastic energy storage of 45.1%+/-6.9% in closer proximity to Freeman and Silver's experimental data. This suggests that although the distance-dependent dielectric implicit solvation approach was favored for its increased speed and decreased computational requirements, an explicit representation of water may be necessary to more accurately model solvent interactions in this particular system. In this paper, we discuss the collagen model described by Freeman and Silver, the present model building approach, the application of the present model to that of Freeman and Silver, and additional assumptions and limitations.

A link between stent radial forces and vascular wall remodeling: the discovery of an optimal stent radial force for minimal vessel restenosis.

Coronary and peripheral artery disease (PAD) continue to be primary causes of morbidity and mortality in western nations; percutaneous transluminal angioplasty (PTA) with stenting has become a popular treatment. Unfortunately, restenosis is a significant problem following intravascular stent placement. This study considers the contribution of stent forces in vascular stenosis and remodeling to develop an equation for identifying the optimal stent force. z-Type stents of three radial forces [low (3.4 N), high (16.4 N), and ultrahigh (19.4 N)] were deployed into the iliac arteries of a juvenile porcine model. Vessel diameters were measured before, after deployment, and again at 30 days. At 30 days animals were killed and the vessels fixed in situ. After implantation, there was a significant increase in total thickness and neointimal hyperplasia with increasing stent force. The model for vessel radius and experimental data was in agreement. The model shows that maximum late-term radius is achieved with a stent deployment stress of 480 kPa, which occurs at the end of the stress-strain curve nonlinear domain and beginning of the high-strain collagen domain. The results and calculations suggest that an optimal stent force exists that is subject to the geometry, structure, and mechanics of the target vessel. To achieve maximum late-term dilatation, stents should not produce stress in the vessel wall greater than the end of the transitional domain of the vessel's stress-strain curve. This finding is extremely important for vascular stent development and will be expanded to preliminary vessel wall injury and atherosclerotic models.

Characterization of a prevascularized biomimetic tissue engineered scaffold for bone regeneration.

Significant bone loss due to disease or severe injury can result in the need for a bone graft, with over 500,000 procedures occurring each year in the United States. However, the current standards for grafting, autografts and allografts, can result in increased patient morbidity or a high rate of failure respectively. An ideal alternative would be a biodegradable tissue engineered graft that fulfills the function of bone while promoting the growth of new bone tissue. We developed a prevascularized tissue engineered scaffold of electrospun biodegradable polymers PLLA and PDLA reinforced with hydroxyapatite, a mineral similar to that found in bone. A composite design was utilized to mimic the structure and function of human trabecular and cortical bone. These scaffolds were characterized mechanically and in vitro to determine osteoinductive and angioinductive properties. It was observed that further reinforcement is necessary for the scaffolds to mechanically match bone, but the scaffolds are successful at inducing the differentiation of mesenchymal stem cells into mature bone cells and vascular endothelial cells. Prevascularization was seen to have a positive effect on angiogenesis and cellular metabolic activity, critical factors for the integration of a graft.

Single-walled carbon nanohorns modulate tenocyte cellular response and tendon biomechanics.

Subfailure ligament and tendon injury remain a significant burden to global healthcare. Here, we present the use of biocompatible single-walled carbon nanohorns (CNH) as a potential treatment for the repair of sub-failure injury in tendons. First, in vitro exposure of CNH to human tenocytes revealed no change in collagen deposition but a significant decrease in cell metabolic activity after 14 days. Additionally, gene expression studies revealed significant downregulation of collagen Types I and III mRNA at 7 days with some recovery after 14 days of exposure. Biomechanical tests with explanted porcine digitorum tendons showed the ability of CNH suspensions to modulate tendon biomechanics, most notably elastic moduli immediately after treatment. in vivo experiments demonstrated the ability of CNH to persist in the damaged matrix of stretch-injured Sprague Dawley rat Achilles tendon but not significantly modify tendon biomechanics after 7 days of treatment. Although these results demonstrate the early feasibility of utility of CNH as a potential modality for tendon subfailure injury, additional work is needed to further validate and ensure clinical efficacy.

Characterization and optimization of a positively charged poly (ethylene glycol)diacrylate hydrogel as an actuating muscle tissue engineering scaffold.

Hydrogels have been used for many applications in tissue engineering and regenerative medicine due to their versatile material properties and similarities to the native extracellular matrix. Poly (ethylene glycol) diacrylate (PEGDA) is an ionic electroactive polymer (EAP), a material that responds to an electric field with a change in size or shape while in an ionic solution, that may be used in the development of hydrogels. In this study, we have investigated a positively charged EAP that can bend without the need of external ions. PEGDA was modified with the positively charged molecule 2-(methacryloyloxy)ethyl-trimethylammonium chloride (MAETAC) to provide its own positive ions. This hydrogel was then characterized and optimized for bending and cellular biocompatibility with C2C12 mouse myoblast cells. Studies show that the polymer responds to an electric field and supports C2C12 viability.

Mechanical and biological evaluation of a hydroxyapatite-reinforced scaffold for bone regeneration.

With over 500,000 bone grafting procedures performed annually in the United States, the advancement of bone regeneration technology is at the forefront of medical research. Many tissue-engineered approaches have been explored to develop a viable synthetic bone graft substitute, but a major challenge is achieving a load-bearing graft that appropriately mimics the mechanical properties of native bone. In this study, sintered hydroxyapatite (HAp) was used to structurally reinforce a scaffold and yield mechanical properties comparable to native bone. HAp was packed into a cylindrical framework and processed under varying conditions to maximize its mechanical properties. The resulting HAp columns were further tested in a 6-week degradation study to determine their physical and mechanical response. The cellular response of sintered HAp was determined using a murine preosteoblast cell line, MC3T3-E1. Cell viability and morphology were studied over a one-week period and MC3T3-E1 differentiation was determined by measuring the alkaline phosphatase levels. Finite element analysis was used to determine the columns' geometric configuration and arrangement within our previously developed composite bone scaffold. It was determined that incorporating four cylindrical HAp columns, fabricated under 44 MPa of pressure and sintered at 1200°C for 5 hr, led to load-bearing properties that match the yield strength of native whole bone. These preliminary results indicate that the incorporation of a mechanically enhanced HAp structural support system is a promising step toward developing one of the first load-bearing bone scaffolds that can also support cell proliferation and osteogenic differentiation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 732-741, 2019.