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.

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.

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.

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 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.

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.

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.

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.

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).

Hydrogel Microencapsulated Insulin-Secreting Cells Increase Keratinocyte Migration, Epidermal Thickness, Collagen Fiber Density, and Wound Closure in a Diabetic Mouse Model of Wound Healing.

Wound healing is a hierarchical process of intracellular and intercellular signaling. Insulin is a potent chemoattractant and mitogen for cells involved in wound healing. Insulin's potential to promote keratinocyte growth and stimulate collagen synthesis in fibroblasts is well described. However, there currently lacks an appropriate delivery mechanism capable of consistently supplying a wound environment with insulin; current approaches require repeated applications of insulin, which increase the chances of infecting the wound. In this study, we present a novel cell-based therapy that delivers insulin to the wound area in a constant or glucose-dependent manner by encapsulating insulin-secreting cells in nonimmunogenic poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We evaluated cell viability and insulin secretory characteristics of microencapsulated cells. Glucose stimulation studies verified free diffusion of glucose and insulin through the microspheres, while no statistical difference in insulin secretion was observed between cells in microspheres and cells in monolayers. Scratch assays demonstrated accelerated keratinocyte migration in vitro when treated with microencapsulated cells. In excisional wounds on the dorsa of diabetic mice, microencapsulated RIN-m cells accelerated wound closure by postoperative day 7; a statistically significant increase over AtT-20ins-treated and control groups. Histological results indicated significantly greater epidermal thickness in both microencapsulated RIN-m and AtT-20ins-treated wounds. The results suggest that microencapsulation enables insulin-secreting cells to persist long enough at the wound site for a therapeutic effect and thereby functions as an effective delivery vehicle to accelerate wound healing.

Poly(3,4-ethylenedioxythiophene) nanoparticle and poly(ɛ-caprolactone) electrospun scaffold characterization for skeletal muscle regeneration.

Injuries to peripheral nerves and/or skeletal muscle can cause scar tissue formation and loss of function. The focus of this article is the creation of a conductive, biocompatible scaffold with appropriate mechanical properties to regenerate skeletal muscle. Poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles (Np) were electrospun with poly(ɛ-caprolactone) (PCL) to form conductive scaffolds. During electrospinning, ribboning, larger fiber diameters, and unaligned scaffolds were observed with increasing PEDOT amounts. To address this, PEDOT Np were sonicated prior to electrospinning, which resulted in decreased conductivity and increased mechanical properties. Multi-walled carbon nanotubes (MWCNT) were added to the 1:2 solution in an effort to increase conductivity. However, the addition of MWCNT had little effect on scaffold conductivity, and the elastic modulus and yield stress of the scaffold increased as a result. Rat muscle cells attached and were active on the 1-10, 1-2, 3-4, and 1-1 PCL-PEDOT scaffolds; however, the 3-4 scaffolds had the lowest level of metabolic activity. Although the scaffolds were cytocompatible, further development of the fabrication method is necessary to produce more highly aligned scaffolds capable of promoting skeletal muscle cell alignment and eventual regeneration.

Cell microencapsulation with synthetic polymers.

The encapsulation of cells into polymeric microspheres or microcapsules has permitted the transplantation of cells into human and animal subjects without the need for immunosuppressants. Cell-based therapies use donor cells to provide sustained release of a therapeutic product, such as insulin, and have shown promise in treating a variety of diseases. Immunoisolation of these cells via microencapsulation is a hotly investigated field, and the preferred material of choice has been alginate, a natural polymer derived from seaweed due to its gelling conditions. Although many natural polymers tend to gel in conditions favorable to mammalian cell encapsulation, there remain challenges such as batch to batch variability and residual components from the original source that can lead to an immune response when implanted into a recipient. Synthetic materials have the potential to avoid these issues; however, historically they have required harsh polymerization conditions that are not favorable to mammalian cells. As research into microencapsulation grows, more investigators are exploring methods to microencapsulate cells into synthetic polymers. This review describes a variety of synthetic polymers used to microencapsulate cells.

Rapid healing of femoral defects in rats with low dose sustained BMP2 expression from PEGDA hydrogel microspheres.

Current strategies for bone regeneration after traumatic injury often fail to provide adequate healing and integration. Here, we combined the poly (ethylene glycol) diacrylate (PEGDA) hydrogel with allogeneic "carrier" cells transduced with an adenovirus expressing BMP2. The system is unique in that the biomaterial encapsulates the cells, shielding them and thus suppressing destructive inflammatory processes. Using this system, complete healing of a 5 mm-long femur defect in a rat model occurs in under 3 weeks, through secretion of 100-fold lower levels of protein as compared to doses of recombinant BMP2 protein used in studies which lead to healing in 2-3 months. New bone formation was evaluated radiographically, histologically, and biomechanically at 2, 3, 6, 9, and 12 weeks after surgery. Rapid bone formation bridged the defect area and reliably integrated into the adjacent skeletal bone as early as 2 weeks. At 3 weeks, biomechanical analysis showed the new bone to possess 79% of torsional strength of the intact contralateral femur. Histological evaluation showed normal bone healing, with no infiltration of inflammatory cells with the bone being stable approximately 1 year later. We propose that these osteoinductive microspheres offer a more efficacious and safer clinical option over the use of rhBMP2.

Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs.

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs.

Cathepsin K-sensitive poly(ethylene glycol) hydrogels for degradation in response to bone resorption.

We propose a new strategy of biomaterial design to achieve selective cellular degradation by the incorporation of cathepsin K-degradable peptide sequences into a scaffold structure so that scaffold biodegradation can be induced at the end of the bone formation process. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels were used as a model biomaterial system in this study. A cathepsin K-sensitive peptide, GGGMGPSGPWGGK (GPSG), was synthesized and modified with acryloyl-PEG-succinimidyl carbonate to produce a cross-linkable cathepsin K-sensitive polymer that can be used to form a hydrogel. Specificity of degradation of the GPSG hydrogels was tested with cathepsin K and proteinase K as a positive control, with both resulting in significant degradation compared to incubation with nonspecific collagenases over a 24-h time period. No degradation was observed when the hydrogels were incubated with plasmin or control buffers. Cell-induced degradation was evaluated by seeding differentiated MC3T3-E1 osteoblasts and RAW264.7 osteoclasts on GPSG hydrogels that were also modified with the cell adhesion peptide RGDS. Resulting surface features and resorption pits were analyzed by differential interference contrast (DIC) and fluorescent images obtained with confocal microscopy. Results from both analyses demonstrated that GPSG hydrogels can be degraded specifically in response to osteoclast attachment but not in response to osteoblasts. In summary, we have demonstrated that by incorporating a cathepsin K-sensitive peptide into a synthetic polymer structure, we can generate biomaterials that specifically respond to cues from the natural process of bone remodeling.

An injectable method for noninvasive spine fusion.

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) induce bone formation but are difficult to localize, and subsequent diffusion from the site of interest and short half-life reduce the efficacy of the protein. Currently, spine fusion requires stripping, decortications of the transverse processes, and an autograft harvest procedure. Even in combination with BMPs, clinical spinal fusion has a high failure rate, presumably because of difficulties in localizing sufficient levels of BMP. PURPOSE: The goal was to achieve reliable spine fusion through a single injection of a cell-based gene therapy system without the need for any surgical intervention. STUDY DESIGN: Eighty-seven immunodeficient (n=44) and immune-competent (n=43) mice were injected along the paraspinous musculature to achieve rapid induction of heterotopic ossification (HO) and ultimately spinal arthrodesis. METHODS: Immunodeficient and immune-competent mice were injected with fibroblasts, transduced with an adenoviral vector to express BMP2, along the paraspinous musculature. Bone formation was evaluated via radiographs, microcomputed tomography, and biomechanical analysis. RESULTS: ew bridging bone between the vertebrae and the fusion to adjacent skeletal bone was obtained as early as 2 weeks. Reduction in spine flexion-extension also occurred as early as 2 weeks after injection of the gene therapy system, with greater than 90% fusion by 4 weeks in all animals regardless of their genetic background. CONCLUSIONS: Injection of our cell-based system into the paraspinous musculature induces spinal fusion that is dependent neither on the cell type nor on the immune status. These studies are the first to harness HO in an immune-competent model as a noninvasive injectable system for clinically relevant spinal fusion and may one day impact human spinal arthrodesis.

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.

Hydrogel microsphere encapsulation of a cell-based gene therapy system increases cell survival of injected cells, transgene expression, and bone volume in a model of heterotopic ossification.

Bone morphogenetic proteins (BMPs) are well known for their osteoinductive activity, yet harnessing this capacity remains a high-priority research focus. We present a novel technology that delivers high BMP-2 levels at targeted locations for rapid endochondral bone formation, enhancing our preexisting cell-based gene therapy system by microencapsulating adenovirus-transduced cells in nondegradable poly(ethylene glycol) diacrylate (PEGDA) hydrogels before intramuscular delivery. This study evaluates the in vitro and in vivo viability, gene expression, and bone formation from transgenic fibroblasts encapsulated in PEGDA microspheres. Fluorescent viability and cytotoxicity assays demonstrated >95% viability in microencapsulated cells. ELISA and alkaline phosphatase assays established that BMP-2 secretion and specific activity from microencapsulated AdBMP2-transduced fibroblasts were not statistically different from monolayer. Longitudinal transgene expression studies of AdDsRed-transduced fibroblasts, followed through live animal optical fluorescent imaging, showed that microencapsulated cells expressed longer than unencapsulated cells. When comparable numbers of microencapsulated AdBMP2-transduced cells were intramuscularly injected into mice, microcomputed tomography evaluation demonstrated that the resultant heterotopic bone formation was approximately twice the volume of unencapsulated cells. The data suggest that microencapsulation protects cells and prolongs and spatially distributes transgene expression. Thus, incorporation of PEGDA hydrogels significantly advances current gene therapy bone repair approaches.

The biomechanical effects of limb lengthening and botulinum toxin type A on rabbit tendon.

Numerous studies have examined the effects of distraction osteogenesis (DO) on bone, but relatively fewer have explored muscle adaptation, and even less have addressed the concomitant alterations that occur in the tendon. The purpose herein was to characterize the biomechanical properties of normal and elongated rabbit (N = 20) tendons with and without prophylactic botulinum toxin type A (BTX-A) treatment. Elastic and viscoelastic properties of Achilles and Tibialis anterior (TA) tendons were evaluated through pull to failure and stress relaxation tests. All TA tendons displayed nonlinear viscoelastic responses that were strain dependent. A power law formulation was used to model tendon viscoelastic responses and tendon elastic responses were fit with a microstructural model. Distraction-elongated tendons displayed increases in compliance and stress relaxation rates over undistracted tendons; BTX-A administration offset this result. The elastic moduli of distraction-lengthened TA tendons were diminished (p = 0.010) when distraction was combined with gastrocnemius (GA) BTX-A administration, elastic moduli were further decreased (p = 0.004) and distraction following TA BTX-A administration resulted in TA tendons with moduli not different from contralateral control (p > 0.05). Compared to contralateral control, distraction and GA BTX-A administration displayed shortened toe regions, (p = 0.031 and 0.038, respectively), while tendons receiving BTX-A in the TA had no differences in the toe region (p > 0.05). Ultimate tensile stress was unaltered by DO, but stress at the transition from the toe to the linear region of the stress-stretch curve was diminished in all distraction-elongated TA tendons (p < 0.05). The data suggest that prophylactic BTX-A treatment to the TA protects some tendon biomechanical properties.