Gulden Camci-Unal, University of Massachusetts Lowell
Josephine Allen, University of Florida
Guillermo Ameer, Northwestern University
Junji Fukuda, Yokohama National University
Acta Biomaterialia (Acta Materialia Inc.) | Elsevier
Acuitive Technologies, Inc.
The Center for Advanced Regenerative Engineering (CARE), Northwestern University
BM04.01: Biomaterials for Regeneration of Tissues I
Tuesday AM, November 27, 2018
Sheraton, 2nd Floor, Independence West
8:00 AM - *BM04.01.01
Bioresorbable Electronic Materials for Wireless Neuroregenerative Therapy
Northwestern University1Show Abstract
Peripheral nerve injuries commonly result in lifelong disability. Even the most advanced surgical procedures and pharmaceutical treatments have limited ability to improve clinical outcomes. Intraoperative electrical stimulation performed at the site of nerve repair is a well-established treatment that can accelerate and improve overall rates of functional recovery. Clinical utilization of electrical stimulation has, however, been limited to the intraoperative period, wherein injured tissue is physically accessible. This talk describes bioresorbable electronic materials and devices that allow for non-pharmacologic neuroregenerative therapy via prolonged post-operative electrical stimulation throughout the healing process, enabling substantially improved outcomes in nerve regeneration and functional recovery compared to the existing intraoperative mode. An essential characteristic of these implantable systems is that they undergo complete dissolution and elimination from the body via natural biochemical processes over timescales matched to operational requirements and without adverse biological effects. The result thereby eliminates the need for secondary surgical extraction and associated risks to the patient and to site of the nerve repair. This type of bioresorbable technology represents a new vehicle for the delivery of non-pharmacologic bioelectric and neuroregenerative therapies in a variety of clinical settings, and a significant paradigm shift in the treatment of critical nerve injuries with limited potential for sensorimotor recovery.
8:30 AM - *BM04.01.02
Elastomeric Polymers for Microfabrication of Organs-on-a-Chip
Univ of Toronto1Show Abstract
Recent advances in human pluripotent stem cell (hPSC) biology enable derivation of essentially any cell type in the human body. However, limitations related to cell maturation, vascularization, cellular fidelity and inter-organ communication still remain.
Here, biological wire (Biowire) technology will be described, developed to specifically enhance maturation levels of hPSC based cardiac tissues, by controlling tissue geometry and electrical field stimulation regime (Nunes et al Nature Methods 2013). We will describe new applications of the Biowire technology in engineering a specifically atrial and specifically ventricular cardiac tissues, safety testing of small molecule kinase inhibitors, potential new cancer drugs, and modelling of left ventricular hypertrophy using patient derived cells.
For probing of more complex physiological questions, dependent on the flow of culture media or blood, incorporation of vasculature is required, most commonly performed in organ-on-a-chip devices. Current organ-on-a-chip devices are limited by the presence of non-physiological materials such as glass and drug-absorbing PDMS as well as the necessity for specialized equipment such as vacuum lines and fluid pumps that inherently limit their throughput. An overview of two new technologies, AngioChip (Zhang et al Nature Materials 2016) and inVADE (Lai et al Advanced Functional Materials 2017) will be presented, that overcome the noted limitations and enable engineering of vascularized liver, vascularized heart tissues and studies of cancer metastasis. These platforms enable facile operation and imaging in a set-up resembling a 96-well plate. Using polymer engineering, we were able to marry two seemingly opposing criteria in these platforms, permeability and mechanical stability, to engineer vasculature suitable for biological discovery and direct surgical anastomosis to the host vasculature.
Finally, to enable minimally invasive delivery of engineered tissues into the body, a new shape-memory scaffold was developed that enables delivery of fully functional tissues on the heart, liver and aorta through a keyhole surgery (Montgomery et al Nature Materials 2017).
9:00 AM - BM04.01.03
Acellular PCL Scaffolds Laden with Fibroblast/Endothelial Cell-Derived Extracellular Matrix for Bone Regeneration
Radoslaw Junka1,Xiaojun Yu1
Stevens Institute of Technology1Show Abstract
Biological scaffolds derived from decellularized tissues function as tissue remodeling templates during bone regeneration. This isolated extracellular matrix (ECM) provides a structural framework that regulates adherence, migration, proliferation, and differentiation of bone residing cells and those in surrounding tissues. Nonetheless, decellularization protocols, like the ones used in isolation of demineralized bone matrix (DBM), require use of acids and other harsh chemicals that render osteoinductive proteins in DBM denatured. Also, lack of vascular cues in DBM presents another impediment to bone healing, and results in non-unions from poor vascularization of the regenerating tissue. To address these limitations, we used tissue engineering approach and tested the regenerative capacity of decellularized ECMs derived from sequential cultures of fibroblasts and endothelial cells grown on polycaprolactone (PCL) fibers. We hypothesized that this vascular ECM would enhance osteoblast proliferation, differentiation, and matrix deposition in vitro. The bottom-up strategy eliminated competition between cell types with varying proliferation rates and allowed for ECM remodeling. ECMs from decellularized cultures were evaluated via methylene and Coomasie blue stains, and their protein and DNA content was quantified. Staining also revealed that endothelial cells grown on fibroblast ECM (Fibro/Endo) form networks resembling capillaries. These structures stained positively for endothelial markers CD31 and vWF. Analysis of SEM images indicated changes in morphology and preferential attachment of endothelial cells to PCL fibers laden with fibroblast ECM. Osteoblasts grown on this Fibro/Endo ECM yielded higher proliferation rates at each time point during 28 day culture. Significantly higher ALP activity in these cultures suggest better capacity of osteoblasts to form bone tissue and higher degree of differentiation. The color area and intensity of Alizarin Red staining of Fibro/Endo cultures revealed uniform and greater calcium deposits than in cultures with only single type of ECM. Relative expression of osteoclacein and osteopontin between culture conditions was compared via immunostaining. Successive culture/decellularization cycles enriched the ECM product, which in turn significantly enhanced proliferation and differentiation of osteoblasts in vitro. Thus, addition of vascular ECM cues to biological scaffolds might lead to improved bone healing rate in vivo. The use of this hybrid ECM can expanded to other combinations with additional cell types, which might further improve regeneration of tissues.
The work was partly supported by the Assistant Secretary of Defense for Health Affairs, through the Peer Reviewed Medical Research Program under Award No. W81XWH-16-1-0132, and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (award number R01EB020640).
9:30 AM -
9:45 AM - BM04.01.06
In Vivo Bioresorbability and Tissue Reaction of Hydroxyapatite/Collagen– (3-Glycidoxypropyl)Trimethoxysilane Injectable Bone Paste
Masanori Kikuchi3,Taira Sato1,Yuki Shirosaki2,Sho Oshima3,4,Yoshihisa Koyama3,Mamoru Aizawa1
Meiji University1,Kyushu Institute of Technology2,National Institute for Materials Science3,Ibaraki University4Show Abstract
Injectable self-setting bone pastes are a user-friendly bone void filler in comparison to dense, porous and granular ones, because pastes are applied for minimal invasive surgeries and are shaped easily to fit bone defects. However, surgeons desire biodegradable bone paste strongly because present bone pastes are very low biodegradable and brittle and a risk to be a cause of secondary bone fracture. Presently available biodegradable bone void fillers in Japan are β-tricalcium phosphates, carbonated apatite and hydroxyapatite/collagen bone-like nanocomposite (HAp/Col). The HAp/Col demonstrates good viscoelasticity and excellent bioresorbability with bone formation ability by incorporating into bone remodeling process. We focused on the HAp/Col as a base material for novel injectable bone cement. In the previous report, the HAp/Col biodegradable self-setting pastes were prepared by a mixing of the HAp/Col powder and aqueous solution of (3-glycidoxypropyl)trimethoxysilane (GPTMS), which is a setting agent by cross-linking of collagen and forming siloxane network by self-condensation. The HAp/Col-GPTMS bone pastes implanted into porcine tibia were resorbed and replaced by newly formed bone within 12 weeks. In this study, the pastes were implanted in the rat tibia and the biological tissue reaction and the absorption behavior of the pastes up to 4 weeks after transplantation were investigated in detail.
The HAp/Col (80/20 in mass ratio) powder at 100 μm or less in particle size was prepared by ball-milling of the HAp/Col compact prepared by a uniaxial pressing after synthesis by the simultaneous titration method. Aqueous solutions of GPTMS as 1.0 and 10 % in volume were prepared by mixing of GPTMS in distilled water followed by 1-hour hydrolyzation of GPTMS. They were mixed at the powder/liquid ratio of 1.00 g/cm3, optimal for the anti-washout property, for 3 min and molded into cylindrical shape with a 2.0 mm in diameter and 2.0 mm in height. The shaped pastes were then incubated for 72 h to be hardened. The hardened pastes were then implanted in 2.0-mm hole defect of proximal SD rat tibia (male, 8 weeks old). At 1, 2 and 4 weeks after the implantation, bioresorption behavior and biological tissue responses of the pastes were investigated by the X-ray µ-computed tomography (µ-CT) and histological observations. All animal tests were authorized by NIMS animal committee (49-2016-3).
The µ-CT observations demonstrated the residual volume of the pastes tended to decrease with the implantation period. The residual volume of the paste with 1.0-% GPTMS significantly decreased between 2 and 4 weeks that is considered to be an osteoclastic resorption. Although no drastically decrease was observed for the paste with 10-% GPTMS, it is expected to be substituted completely with newly formed bone deduced from the results of pig test. In addition, the results of histological observations will be presented on a podium.
10:30 AM - *BM04.01.07
Bioactive Microrods for the Attenuation of Chronic Cardiac Fibrosis
Long V. Le1,Priya Mohindra1,Qizhi Fang2,Rich Sievers2,Michael Mkrtschjan3,Brenda Russell3,Randall Lee2,Tejal Desai4,1
UC Berkeley-UCSF Graduate Group in Bioengineering1,University of California, San Francisco2,University of Illinois at Chicago3,UCSF Bioengineering and Therapeutic Sciences4Show Abstract
Coronary artery disease is the leading cause of death in the United States, with over 700,000 myocardial infarctions (MI) occurring each year. While effective strategies have been developed to reduce mortality rates from the acute event, there remain significant challenges to preventing the formation of fibrous scar tissue, which leads to reduced cardiac function and eventual heart failure. Here, we demonstrate the fabrication of hyaluronic acid (HA)-based microrods with tunable size, shape, and stiffness for the attenuation of chronic cardiac fibrosis. These microrods modify the mechanical properties of the tissue microenvironment and modulate the fibrotic phenotype through mechanotransduction pathways. Additionally, HA is a naturally occurring material that is bioresorbable and exhibits several wound healing properties, making it a promising material for this microtopography-based approach. We show that fibroblasts closely interact with these microrods in vitro and in vivo, leading to dramatic changes in proliferation, collagen expression and fibrotic phenotype. NIH-3T3s and primary NVRFs attached to HA microrods and often conform to the rod geometry. When grown in the presence of HA microrods, fibroblasts have reduced expression of collagen I and aSMA, suggesting that HA microrods may be able to attenuate the conversion of fibroblasts to the active myofibroblast that is responsible for the overproduction of scar tissue. When injected into the myocardium of a rat infarct model, HA microrods improved cardiac function at 6 weeks post infarct. We have previously shown that PEG-based microrods are able to decrease the loss of cardiac function caused by MI, increasing the % change in EF from -11% to -3%. Impressively, HA-based microrods are able to increase EF by nearly +10%, suggesting that the rods are not only able to reduce deterioration of function, but restore function to the injured heart as well. This may be due to HA being bioresorbable and able to elicit additional biochemical effects through HA signaling pathways as elucidated on our studies. We also show demonstrate that the biochemical conjugation of the microrods with angiogenic peptides can further enhance the ability of this material to promote tissue regeneration. Such materials based approaches can be used in a variety of disease settings including vascular and musculoskeletal, opening up new therapeutic approaches for tissue regeneration.
11:00 AM - *BM04.01.08
Platform Technologies for Engineering Functional Composite Tissues
Johns Hopkins University1Show Abstract
Regenerative Engineering strategies have unprecedented potential to restore function to large musculoskeletal injuries. The talk will be used to describe methods developed together with collaborators to treat critical-sized segmental bone defects and volumetric muscle loss. Our lab has developed promising 3D-printing scaffold technologies that combine poly-ε-caprolactone (PCL) with decellularized bone (DCB) matrix to make composite PCL-DCB scaffolds. We have demonstrated the osteoinductive properties of PCL-DCB scaffolds by assessing the de novo bone formation when seeded with adipose-derived stromal/stem cells and stromal vascular fraction (SVF) and implanted in murine critical-sized cranial defects. Murine models of critical-sized bone defects are also useful in understanding the role of vascularization in bone regeneration. We have recently demonstrated its use in the application of a novel multimodality imaging platform for acquiring in vivo images of microvascular architecture, microvascular blood flow and tracer/cell tracking via intrinsic optical signaling, laser speckle contrast and fluorescence imaging. We are currently using these techniques to explore the impact of rapid, early-stage revascularization on bone healing. In considering translation to humans, however, the complex loading regimens in the craniofacial skeleton make the design of clinically applicable scaffolds inherently more complex than those used to treat murine calvariae. To address this, we are developing algorithms for designing scaffolds with heterogenous porous structures tailored to the expected physiologic loads.
To treat volumetric muscle loss, our team is developing a novel biomaterial-based approach to overcome scarring and induce regeneration of densely vascularized muscle in non-healing, skeletal muscle defects. In particular, we have demonstrated the capacity of novel electrospun fibrin hydrogel scaffolds seeded with myoblasts to regenerate the structure and function of damaged muscle. Myoblast-seeded scaffolds enabled remarkable muscle regeneration with high myofiber and vascular densities after 2 and 4 weeks, mimicking that of native skeletal muscle (while acellular scaffolds lacked muscle regeneration). Both myoblast-seeded and acellular scaffolds fully recovered muscle contractile function to uninjured values after 2 and 4 weeks. In ongoing studies, we are evaluating the potential for tuning the myogenic impact by varying the mechanical properties of the scaffolds. I will also describe our ongoing work on modulating the biochemical characteristics of the scaffold to stimulate neural infiltration and the formation of functional neuromuscular junctions within regenerated skeletal muscle tissues.
11:30 AM - BM04.01.09
Tissue Origami for Template-Guided Mineralization
University of Massachusetts Lowell1Show Abstract
Due to disease, degeneration, trauma, and aging, bone loss occurs in the body. Although there have been remarkable improvements in development of functional bone scaffolds, it remains difficult to fabricate porous and biocompatible constructs in physiologically relevant sizes (cm-scale). Herein we developed biomineralized origami-inspired paper scaffolds in three-dimensions (3D). To our knowledge, this work is the first demonstration that paper can be used as a 3D construct to induce template-guided mineralization by osteoblasts.
In this work, we used the principles of origami to fabricate free-standing paper scaffolds in cm-scale. Because paper is an extremely flexible material that can easily be cut, creased, and folded to form 3D structures, the scaffolds were easily fabricated in a variety of different geometries. This feature can potentially be useful in generation of constructs for patient-specific applications especially for patients who have defects of irregular sizes and shapes. After sterilizing the constructs, they were seeded with osteoblasts in a collagen matrix. The samples were cultured up to 21 days and mineralization was evaluated using various assays including colorimetric assays, immunocytochemistry, high-resolution imaging (SEM), and micro-computed tomography (micro-CT). We also performed in vivo subcutaneous implantation experiments in a rat model.
In this project, we generated paper scaffolds in different shapes, sizes, and configurations (mm-cm scale). Due to its porous structure, paper allowed for transport of oxygen and nutrients across its thickness. Paper scaffolds supported a homogenous distribution of cells within their 3D structures. In our experiments, proliferation of osteoblasts increased until day three and then decreased. Hydroxyapatite content of the samples indicated that there was a progressive increase in the amount of hydroxyapatite in the paper scaffolds over 21 day of culture period. We used SEM to visualize the deposition of mineral clusters, and EDAX to calculate the ratio of calcium to phosphate. Our in vivo experiments demonstrated that paper scaffolds did not cause inflammation. The paper implants integrated with the existing tissue strongly and rapidly vascularized.
To sum up, we have shown that origami-inspired tissue engineering is useful for template-guided mineralization. We obtained partially mineralized scaffolds in various 3D geometries. The osteoblasts deposited calcium phosphate in these scaffolds and induced template-guided mineralization. Our approach used paper, a readily available material, as the cell culture scaffold. Paper has great potential to tackle the limitations of traditional scaffolds