The effect of polymer molecular weight and cell seeding density on viability of cells entrapped within PEGDA hydrogel microspheres.

Cell microencapsulation can be used in tissue engineering as a scaffold or physical barrier that provides immunoisolation for donor cells. When used as a barrier, microencapsulation shields donor cells from the host immune system when implanted for cell therapies. Maximizing therapeutic product delivery per volume of microencapsulated cells necessitates first optimising the viability of entrapped cells. Although cell microencapsulation within alginate is well described, best practices for cell microencapsulation within polyethylene glycol is still being elucidated. In this study we microencapsulate mouse preosteoblast cells within polyethylene glycol diacrylate (PEGDA) hydrogel microspheres of varying molecular weight or seeding densities to assess cell viability in relation to cell density and polymer molecular weight. Diffusion studies revealed molecule size permissible by each molecular weight PEGDA towards correlating viability with polymer mesh size. Results demonstrated higher cell viability in higher molecular weight PEGDA microspheres and when cells were seeded at higher cell densities.

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.

Cell-based therapies for spinal fusion.

During spinal fusion procedures, bone grafts are placed to promote bone healing and to provide stability. The autologous graft is the current clinical standard of care due to its ability to initiate bone formation and because it poses no risk of rejection; however, it has drawbacks such as donor site morbidity and limited supply. Due to processing for sterility and storage, allogeneic grafts have reduced osteoinductive properties and thus must be delivered with osteoinductive agents. As a result, bone morphogenetic proteins have been used increasingly to augment bone repair, but in certain locations these proteins can cause complications such as swelling and ectopic bone formation. The drawbacks associated with these treatments have prompted increased investigations into using cells to deliver osteoinductive agents. Clinical studies have demonstrated that when osteoprogenitor cells are combined with osteoconductive materials, fusion rates are comparable to autograft results. Preclinical investigations have achieved superior spinal fusion rates in as little as two weeks using cells genetically modified to deliver osteoinductive agents. Immunoisolation of allogeneic cells by microencapsulation has demonstrated the feasibility of using non-autologous cells, thereby eliminating the need for immunosuppressants. This chapter describes the latest research advances in promoting spinal fusion using these cell-based therapies.

Cell-based gene therapy for repair of critical size defects in the rat fibula.

More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies.

Distraction osteogenesis-induced muscle fibrosis may not be associated with TGF-ß1.

BACKGROUND: Transforming growth factor-ß 1 (TGF-ß1) participates in the synthesis and deposition of collagen. It has been implicated in fibrosis of tendons in wound-healing models but has never been studied in muscles with respect to distraction osteogenesis. METHODS: Using a rabbit model of distraction osteogenesis, we distracted the left tibias of 36 New Zealand white rabbits at 0.75 mm/d for 20 days. To determine whether suramin, an antagonist of TGF-ß, could aid in the prevention of fibrosis, we injected it into the anterior tibialis muscle [12 rabbits received low-dose suramin (50 mg), 12 received high-dose suramin (100 mg), and 12 received sham injections]. Half of each group was killed at the end of distraction (day 24) and the other half at day 60. At the time of killing the rabbits, joint range of motion was measured, and strength and morphometric measures of the muscle were taken. Muscle was harvested and immunolabeled for TGF-ß1. All findings were compared between study limbs and control (right) limbs. RESULTS: The comparison failed to demonstrate improvements in the range of motion, and in strength or morphometric muscle development. Immunolabeling for TGF-ß1 failed to show any staining in the intramuscular fibrosis. Paradoxically, muscle injected with high-dose suramin had the highest degree of fibrosis. CONCLUSIONS: We conclude that TGF-ß1 may not be the primary mediator of muscle fibrosis in distraction osteogenesis. CLINICAL RELEVANCE: Injection of suramin may not prevent contracture formation after distraction osteogenesis.

Biomaterials for human space exploration: A review of their untapped potential.

As biomaterial advances make headway into lightweight radiation protection, wound healing dressings, and microbe resistant surfaces, a relevance to human space exploration manifests itself. To address the needs of the human in space, a knowledge of the space environment becomes necessary. Both an understanding of the environment itself and an understanding of the physiological adaptations to that environment must inform design parameters. The space environment permits the fabrication of novel biomaterials that cannot be produced on Earth, but benefit Earth. Similarly, designing a biomaterial to address a space-based challenge may lead to novel biomaterials that will ultimately benefit Earth. This review describes several persistent challenges to human space exploration, a variety of biomaterials that might mitigate those challenges, and considers a special category of space biomaterial. STATEMENT OF SIGNIFICANCE: This work is a review of the major human and environmental challenges facing human spaceflight, and where biomaterials may mitigate some of those challenges. The work is significant because a broad range of biomaterials are applicable to the human space program, but the overlap is not widely known amongst biomaterials researchers who are unfamiliar with the challenges to human spaceflight. Additionaly, there are adaptations to microgravity that mimic the pathology of certain disease states ("terrestrial analogs") where treatments that help the overwhelmingly healthy astronauts can be applied to help those with the desease. Advances in space technology have furthered the technology in that field on Earth. By outlining ways that biomaterials can promote human space exploration, space-driven advances in biomaterials will further biomaterials technology.

Micropatterning biomineralization with immobilized mother of pearl proteins.

In response to the drawbacks of autograft donor-site morbidity and bone morphogenetic protein type 2 (BMP2) carcinogenesis and ectopic bone formation, there has been an increased research focus towards developing alternatives capable of achieving spatial control over bone formation. Here we show for the first time both osteogenic differentiation and mineralization (from solution or mediated by cells) occurring within predetermined microscopic patterns. Our results revealed that both PEGylated BMP2 and nacre proteins induced stem cell osteodifferentiation in microscopic patterns when these proteins were covalently bonded in patterns onto polyethylene glycol diacrylate (PEGDA) hydrogel substrates; however, only nacre proteins induced mineralization localized to the micropatterns. These findings have broad implications on the design and development of orthopedic biomaterials and drug delivery.

Development of an Electroactive Hydrogel as a Scaffold for Excitable Tissues.

For many cells used in tissue engineering applications, the scaffolds upon which they are seeded do not entirely mimic their native environment, particularly in the case of excitable tissues. For instance, muscle cells experience contraction and relaxation driven by the electrical input of an action potential. Electroactive materials can also deform in response to electrical input; however, few such materials are currently suitable as cell scaffolds. We previously described the development of poly(ethyelene glycol) diacrylate-poly(acrylic acid) as an electroactive scaffold. Although the scaffold itself supported cell growth and attachment, the voltage (20 V) required to actuate these scaffolds was cytotoxic. Here, we describe the further development of our hydrogels into scaffolds capable of actuation at voltages (5 V) that were not cytotoxic to seeded cells. This study describes the critical next steps towards the first functional electroactive tissue engineering scaffold.

Calcein Binding to Assess Mineralization in Hydrogel Microspheres.

We describe a method to assess mineralization by osteoblasts within microspheres using calcein. Fluorescence imaging of calcein bound to the calcium in hydroxyapatite permits assessment of the mineralized portion of the extracellular matrix. Colorimetric imaging of Alizarin Red S complexed with calcium also gives measures of mineralization, and in tissue cultures calcein and Alizarin Red S have been shown to bind to the same regions of mineral deposits. We show that when the mineralization takes place within hydrogel microspheres, Alizarin Red S does not stain mineral deposits as consistently as calcein. As tissue engineers increasingly encapsulate osteoprogenitors within hydrogel scaffolds, calcein staining may prove a more reliable method to assess this mineralization.

Spatiotemporal Control Strategies for Bone Formation through Tissue Engineering and Regenerative Medicine Approaches.

Global increases in life expectancy drive increasing demands for bone regeneration. The gold standard for surgical bone repair is autografting, which enjoys excellent clinical outcomes; however, it possesses significant drawbacks including donor site morbidity and limited availability. Although collagen sponges delivered with bone morphogenetic protein, type 2 (BMP2) are a common alternative or supplement, they do not efficiently retain BMP2, necessitating extremely high doses to elicit bone formation. Hence, reports of BMP2 complications are rising, including cancer promotion and ectopic bone formation, the latter inducing complications such as breathing difficulties and neurologic impairments. Thus, efforts to exert spatial control over bone formation are increasing. Several tissue engineering approaches have demonstrated the potential for targeted and controlled bone formation. These approaches include biomaterial scaffolds derived from synthetic sources, e.g., calcium phosphates or polymers; natural sources, e.g., bone or seashell; and immobilized biofactors, e.g., BMP2. Although BMP2 is the only protein clinically approved for use in a surgical device, there are several proteins, small molecules, and growth factors that show promise in tissue engineering applications. This review profiles the tissue engineering advances in achieving control over the location and onset of bone formation (spatiotemporal control) toward avoiding the complications associated with BMP2.

Coencapsulation of ISCs and MSCs Enhances Viability and Function of both Cell Types for Improved Wound Healing.

INTRODUCTION: We previously demonstrated that insulin secreting cells (ISCs) accelerate healing of chronic wounds, and it is known that mesenchymal stem cells (MSCs) also accelerate wound healing. Here, we report that the combination of both cell types coencapsulated into a synthetic hydrogel dressing accelerates chronic wound healing 3 × faster than control and 2 × faster than each cell type delivered singly. Specifically, insulin released by ISCs activates the PI3/Akt pathway, which is vital to the function and survival of MSCs. MSCs in turn improve the viability and function of ISCs. MATERIALS AND METHODS: MSCs and/or rat islet tumor RIN-m cells were encapsulated into polyethylene glycol diacrylate hydrogel sheets and applied to 1 cm2 full thickness excisional wounds on the dorsa of genetically diabetic male mice (BKS.Cg-m +/+Leprdb/J) in accordance with protocols approved by the Rutgers IACUC. Encapsulated cell viability was assessed using a LIVE/DEAD® Viability/Cytotoxicity Kit. Akt phosphorylation, insulin, VEGF, and TGF-ß1 secretion were assessed by ELISA. Animals were sacrificed on postoperative days 14 and 28 and wound tissue was collected for histological and western blot analysis. RESULTS: ISC:MSC combination groups had the highest levels of every secreted product and phosphorylated Akt, and closed wounds in 14 days, ISC-only or MSC-only groups closed wounds in 28 days, control groups closed wounds in 40 days. Further, ISC:MSC groups healed without intermediate scab or scar. CONCLUSIONS: Combining MSCs with ISCs results in a more robust healing response than singly delivered cells, warranting further investigation of coencapsulation for MSC therapies.

Strain and Vibration in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into any mesenchymal tissue, including bone, cartilage, muscle, and fat. MSC differentiation can be influenced by a variety of stimuli, including environmental and mechanical stimulation, scaffold physical properties, or applied loads. Numerous studies have evaluated the effects of vibration or cyclic tensile strain on MSCs towards developing a mechanically based method of differentiation, but there is no consensus between studies and each investigation uses different culture conditions, which also influence MSC fate. Here we present an overview of the response of MSCs to vibration and cyclic tension, focusing on the effect of various culture conditions and strain or vibration parameters. Our review reveals that scaffold type (e.g., natural versus synthetic; 2D versus 3D) can influence cell response to vibration and strain to the same degree as loading parameters. Hence, in the efforts to use mechanical loading as a reliable method to differentiate cells, scaffold selection is as important as method of loading.

Activin A improves retinal pigment epithelial cell survival on stiff but not soft substrates.

In several retinal degenerative disease pathologies, such as dry age-related macular degeneration (AMD), the retinal pigment epithelium (RPE) cell monolayer becomes dysfunctional. Promising tissue engineering treatment approaches implant RPE cells on scaffolds into the subretinal space. However, these approaches are not without challenges. Two major challenges that must be addressed are RPE dedifferentiation and the inflammatory response to cell/scaffold implantation. Design and optimization of scaffold cues for the purpose of RPE transplantation remain relatively unexplored, specifically the mechanical properties of the scaffolds. Prior work from our group indicated that by varying substrate moduli significant differences could be induced in cell cytoskeleton structure, cellular activity, and expression of inflammatory markers. We hypothesized that Activin A would provide rescue effects for cells demonstrating dedifferentiated characteristics. Results demonstrated that for cells on low modulus scaffolds, the mechanical environment was the dominating factor and Activin A was unable to rescue these cells. However, Activin A did demonstrate rescue effects for cells on high modulus scaffolds. This finding indicates that when cultured on scaffolds with an appropriate modulus, exogenous factors, such as Activin A, can improve RPE cell expression, morphology, and activity, while an inappropriate scaffold modulus can have devastating effects on RPE survival regardless of chemical stimulation. These findings have broad implications for the design and optimization of scaffolds for long-term successful RPE transplantation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2871-2880, 2018.

The effect of low-magnitude, high-frequency vibration on poly(ethylene glycol)-microencapsulated mesenchymal stem cells.

Low-magnitude, high-frequency vibration has stimulated osteogenesis in mesenchymal stem cells when these cells were cultured in certain types of three-dimensional environments. However, results of osteogenesis are conflicting with some reports showing no effect of vibration at all. A large number of vibration studies using three-dimensional scaffolds employ scaffolds derived from natural sources. Since these natural sources potentially have inherent biochemical and microarchitectural cues, we explored the effect of low-magnitude, high-frequency vibration at low, medium, and high accelerations when mesenchymal stem cells were encapsulated in poly(ethylene glycol) diacrylate microspheres. Low and medium accelerations enhanced osteogenesis in mesenchymal stem cells while high accelerations inhibited it. These studies demonstrate that the isolated effect of vibration alone induces osteogenesis.

Biomanufacturing for clinically advanced cell therapies.

The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.

Effects of botulinum toxin A on functional outcome during distraction osteogenesis.

Distraction osteogenesis is useful for correcting limb length inequality, deformities, or short stature. Despite success with bone formation, soft tissue maladaptations including muscle and joint contracture may lead to undesirable results. Botulinum toxin A has been useful in treating spasticity in cerebral palsy, and has been used clinically in select cases to allay contracture in distraction osteogenesis. This study examines the toxin's efficacy in preventing distraction-induced loss of muscle strength and range of motion. The left tibias of 15 New Zealand White rabbits were distracted 1.5 mm/day until approximately a 20% gain was achieved. Each treatment group was divided into animals injected with saline or botulinum toxin in either the gastrocnemius or tibialis anterior muscles. A control group of two additional animals underwent no surgical procedure. Strength and range of motion were assessed prior to, and following, the experiment. At the study's end, animals were euthanized and muscles were harvested, when lengths and weights were recorded. All muscles injected with botulinum toxin showed decreased wet weight and persistent weakness upon completion of the study. Range of motion decreased in all distracted animals. When the gastrocnemius was injected, its strength was reduced but the tibialis anterior strength was preserved, and the limb achieved 22% greater dorsiflexion than saline controls (p = 0.016). When the tibialis anterior received the toxin, plantarflexion was increased by 23% (p = 0.049). Botulinum toxin injection prior to limb distraction increases the "post-lengthened" excursion of the injected muscle and this increased length may have a protective effect on its antagonist. In toxin-injected gastrocnemius muscles, the level of equinus contracture is reduced due to length gains in the Achilles tendon while the anterior tibialis maintains its ability to generate torque. Injection of botulinum toxin in the gastrocnemius may minimize equinus contracture and protect the anterior tibialis from damage during human tibial lengthening. Longer follow-up studies are needed to ensure that toxin-induced muscle weakness resolves with time.

Polymeric Materials for Cell Microencapsulation.

Mammalian cells have been microencapsulated within both natural and synthetic polymers for over half a century. Specifically, in the last 36 years microencapsulated cells have been used therapeutically to deliver a wide range of drugs, cytokines, growth factors, and hormones while enjoying the immunoisolation provided by the encapsulating material. In addition to preventing immune attack, microencapsulation prevents migration of entrapped cells. Cells can be microencapsulated in a variety of geometries, the most common being solid microspheres and hollow microcapsules. The micrometer scale permits delivery by injection and is within diffusion limits that allow the cells to provide the necessary factors that are missing at a target site, while also permitting the exchange of nutrients and waste products. The majority of cell microencapsulation is performed with alginate/poly-L-lysine microspheres. Since alginate itself can be immunogenic, for cell-based therapy applications various groups are investigating synthetic polymers to microencapsulate cells. We describe a protocol for the formation of microspheres and microcapsules using the synthetic polymer poly(ethylene glycol) diacrylate (PEGDA).

The influence of substrate modulus on retinal pigment epithelial cells.

Although transplantation of retinal pigment epithelial (RPE) cells has shown promise for the treatment of retinal degenerative diseases, this therapeutic approach is not without challenges. Two major challenges are RPE cell dedifferentiation and inflammatory response following transplantation. The aim of this work is to understand how the rigidity of a scaffold, a relatively unexplored design aspect in retinal tissue engineering, affects RPE cells, particularly the pathways associated with the aforementioned challenges. Poly(ethylene glycol) diacrylate (PEGDA) of varying molecular weights from 3.4 to 20 kDa were photopolymerized to fabricate scaffolds. The Young's modulus of the scaffolds varied from 60 to 1200 kPa. A cell study was then conducted to test the effects of scaffold rigidity on RPE cells. A cell adhesion peptide motif of arginine-glycine-aspartic acid-serine (RGDS) was conjugated to 60 and 1200 kPa scaffolds and ARPE-19 cells, a human RPE cell line, were seeded onto these hydrogels. Cells grown on scaffolds demonstrated qualitatively different adhesion properties, metabolic activity, and gene expression at an mRNA level. IL-6 and MCP-1, two inflammation markers known to recruit microglial into the retina, had the same expression pattern with cells having the highest expression on the high modulus scaffold and lowest expression on the control substrate. This study demonstrates that scaffold rigidity, an important design parameter in other areas of tissue engineering, affects cell adhesion, activity, and expression of RPE cells. Though more exploration is needed, this begins to lay a foundation for optimizing scaffold rigidity to promote long-term success of RPE scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1260-1266, 2017.

Scaffolds for retinal pigment epithelial cell transplantation in age-related macular degeneration.

In several retinal degenerative diseases, including age-related macular degeneration, the retinal pigment epithelium, a highly functionalized cell monolayer, becomes dysfunctional. These retinal diseases are marked by early retinal pigment epithelium dysfunction reducing its ability to maintain a healthy retina, hence making the retinal pigment epithelium an attractive target for treatment. Cell therapies, including bolus cell injections, have been investigated with mixed results. Since bolus cell injection does not promote the proper monolayer architecture, scaffolds seeded with retinal pigment epithelium cells and then implanted have been increasingly investigated. Such cell-seeded scaffolds address both the dysfunction of the retinal pigment epithelium cells and age-related retinal changes that inhibit the efficacy of cell-only therapies. Currently, several groups are investigating retinal therapies using seeded cells from a number of cell sources on a variety of scaffolds, such as degradable, non-degradable, natural, and artificial substrates. This review describes the variety of scaffolds that have been developed for the implantation of retinal pigment epithelium cells.

Carotid endarterectomy in octogenarian veterans: does age affect outcome? A single-center experience.

BACKGROUND: The efficacy of carotid endarterectomy (CEA) in octogenarians is controversial. Recent reports have examined this question in the general population, but little data exist on veterans. With the emergence of carotid artery stenting, we need to evaluate the role of CEA in treating elderly veterans with carotid stenosis. METHODS: Retrospective chart review of all CEAs performed between January 1995 and December 2004. RESULTS: A total of 286 procedures were performed in 239 patients; 39 procedures were performed in 33 octogenarians, and 247 procedures were performed in 206 younger veterans. Both groups had similar preoperative comorbidities. There were no statistically significant differences between octogenarians and younger veterans for postoperative stroke (2% vs. 1%), death (0% vs. 1%), myocardial infarction (5% vs. 2%), length of stay (7 +/- 19 vs. 3 +/- 8 days), or 4-year survival (53% vs. 57%). CONCLUSIONS: CEA can be safely performed in octogenarian veterans with outcomes similar to younger veterans.