Fabrication and Characterization of Quad-Component Bioinspired Hydrogels to Model Elevated Fibrin Levels in Central Nervous Tissue Scaffolds.

Multicomponent interpenetrating polymer network (mIPN) hydrogels are promising tissue-engineering scaffolds that could closely resemble key characteristics of native tissues. The mechanical and biochemical properties of mIPNs can be finely controlled to mimic key features of target cellular microenvironments, regulating cell-matrix interactions. In this work, we fabricated hydrogels made of collagen type I (Col I), fibrin, hyaluronic acid (HA), and poly (ethylene glycol) diacrylate (PEGDA) using a network-by-network fabrication approach. With these mIPNs, we aimed to develop a biomaterial platform that supports the in vitro culture of human astrocytes and potentially serves to assess the effects of the abnormal deposition of fibrin in cortex tissue and simulate key aspects in the progression of neuroinflammation typically found in human pathologies such as Alzheimer's disease (AD), Parkinson's disease (PD), and tissue trauma. Our resulting hydrogels closely resembled the complex modulus of AD human brain cortex tissue (~7.35 kPa), promoting cell spreading while allowing for the modulation of fibrin and hyaluronic acid levels. The individual networks and their microarchitecture were evaluated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Human astrocytes were encapsulated in mIPNs, and negligible cytotoxicity was observed 24 h after the cell encapsulation.

A Bioinspired Astrocyte-Derived Coating Promotes the In Vitro Proliferation of Human Neural Stem Cells While Maintaining Their Stemness.

The repair of neuronal tissue is a challenging process due to the limited proliferative capacity of neurons. Neural stem cells (NSCs) can aid in the regeneration process of neural tissue due to their high proliferation potential and capacity to differentiate into neurons. The therapeutic potential of these cells can only be achieved if sufficient cells are obtained without losing their differentiation potential. Toward this end, an astrocyte-derived coating (HAc) was evaluated as a promising substrate to promote the proliferation of NSCs. Mass spectroscopy and scanning electron microscopy were used to characterize the HAc. The proliferation rate and the expression of stemness and differentiation markers in NSCs cultured on the HAc were evaluated and compared to the responses of these cells to commonly used coating materials including Poly-L-Ornithine (PLO), and a Human Induced Pluripotent Stem Cell (HiPSC)-based coating. The use of the HAc promotes the in vitro cell growth of NSCs. The expression of the stemness markers Sox2 and Nestin, and the differentiation marker DCX in the HAc group was akin to the expression of these markers in the controls. In summary, HAc supported the proliferation of NSCs while maintaining their stemness and neural differentiation potential.

Growth Factor Binding Peptides in Poly (Ethylene Glycol) Diacrylate (PEGDA)-Based Hydrogels for an Improved Healing Response of Human Dermal Fibroblasts.

Growth factors (GF) are critical cytokines in wound healing. However, the direct delivery of these biochemical cues into a wound site significantly increases the cost of wound dressings and can lead to a strong immunological response due to the introduction of a foreign source of GFs. To overcome this challenge, we designed a poly(ethylene glycol) diacrylate (PEGDA) hydrogel with the potential capacity to sequester autologous GFs directly from the wound site. We demonstrated that synthetic peptide sequences covalently tethered to PEGDA hydrogels physically retained human transforming growth factor beta 1 (hTGFß1) and human vascular endothelial growth factor (hVEGF) at 3.2 and 0.6 ng/mm2, respectively. In addition, we demonstrated that retained hTGFß1 and hVEGF enhanced human dermal fibroblasts (HDFa) average cell surface area and proliferation, respectively, and that exposure to both GFs resulted in up to 1.9-fold higher fraction of area covered relative to the control. After five days in culture, relative to the control surface, non-covalently bound hTGFß1 significantly increased the expression of collagen type I and hTGFß1 and downregulated vimentin and matrix metalloproteinase 1 expression. Cumulatively, the response of HDFa to hTGFß1 aligns well with the expected response of fibroblasts during the early stages of wound healing.

Modeling the effects of hyaluronic acid degradation on the regulation of human astrocyte phenotype using multicomponent interpenetrating polymer networks (mIPNs).

Hyaluronic acid (HA) is a highly abundant component in the extracellular matrix (ECM) and a fundamental element to the architecture and the physiology of the central nervous system (CNS). Often, HA degradation occurs when an overreactive inflammatory response, derived from tissue trauma or neurodegenerative diseases such as Alzheimer's, causes the ECM in the CNS to be remodeled. Herein, we studied the effects of HA content as a key regulator of human astrocyte (HAf) reactivity using multicomponent interpenetrating polymer networks (mIPNs) comprised of Collagen I, HA and poly(ethylene glycol) diacrylate. The selected platform facilities the modulation of HA levels independently of matrix rigidity. Total astrocytic processes length, number of endpoints, the expression of the quiescent markers: Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1) and Glutamate Aspartate Transporter (GLAST); the reactive markers: Glial Fibrillary Acidic Protein (GFAP) and S100 Calcium-Binding Protein ß (S100ß); and the inflammatory markers: Inducible Nitric Oxide Synthase (iNOS), Interleukin 1ß (IL-1ß) and Tumor Necrosis Factor Alpha (TNFα), were assessed. Cumulatively, our results demonstrated that the decrease in HA concentration elicited a reduction in the total length of astrocytic processes and an increase in the expression of HAf reactive and inflammatory markers.

In vitro evaluation of anti-fibrotic effects of select cytokines for vocal fold scar treatment.

Scarring of the vocal fold lamina propria (LP) can cause considerable voice disorders due to reduced pliability in scar tissue, attributed in part to abnormal extracellular matrix (ECM) deposition produced by the fibrotic vocal fold fibroblast (fVFF). Cytokines with anti-fibrotic potential have been investigated to limit abnormal LP ECM, but are limited by the need for repeat injections. Moreover, the potentially significant role played by activated macrophages (AMOs) is usually not considered even though the interaction between AMO and fibrotic fibroblasts is known to regulate scar formation across different tissues. AMO are also regulated by cytokines that are used for LP scar removal, but little is known about AMO behaviors in response to these cytokines within the context of LP scar. In the present study, we evaluated anti-fibrotic effects of hepatocyte growth factor (HGF), interleukin-10 (IL-10) and interleukin-6 (IL-6) in a 3D, in vitro fVFF-AMO co-culture system using poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Data from all cytokines was synthesized into a heat-map that enabled assessment of specific associations between AMO and fVFF phenotypes. Cumulatively, our results indicated that both HGF and IL-10 are potentially anti-fibrotic (reduction in fibrotic markers and enhancement in normal, anti-fibrotic VFF markers), while IL-6 displays more complex, marker specific effects. Possible associations between AMO and fVFF phenotypes were found and may highlight a potential desirable macrophage phenotype. These data support the therapeutic potential of HGF and IL-10 for LP scar treatment, and shed light on future strategies aimed at targeting specific AMO phenotypes. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1056-1067, 2019.

Collagen Based Multicomponent Interpenetrating Networks as Promising Scaffolds for 3D Culture of Human Neural Stem Cells, Human Astrocytes, and Human Microglia.

This work describes for the first time the fabrication and characterization of multicomponent interpenetrating networks composed of collagen I, hyaluronic acid, and poly(ethylene glycol) diacrylate for the 3D culture of human neural stem cells, astrocytes, and microglia. The chemical composition of the scaffolds can be modulated while maintaining values of complex moduli within the range of the mechanical performance of brain tissue (∼6.9 kPa) and having cell viability exceeding 84%. The developed scaffolds are a promising new family of biomaterials that can potentially serve as 3D in vitro models for studying the physiology and physiopathology of the central nervous system.

Refined assessment of the impact of cell shape on human mesenchymal stem cell differentiation in 3D contexts.

Numerous studies have demonstrated that the differentiation potential of human mesenchymal stem cells (hMSCs) can be modulated by chemical and physical cues. In 2D contexts, inducing different cell morphologies, by varying the shape, area and/or curvature of adhesive islands on patterned surfaces, has significant effects on hMSC multipotency and the onset of differentiation. In contrast, in vitro studies in 3D contexts have suggested that hMSC differentiation does not directly correlate with cell shape. However, in 3D, the effects of cell morphology on hMSC differentiation have not yet been clearly established due to the chemical and physical properties being intertwined in 3D matrices. In this work, we studied the effects of round or elongated cell morphologies on hMSC differentiation independently of scaffold composition, modulus, crosslink density and cell-mediated matrix remodeling. The effects of cell shape on hMSC lineage progression were studied using three different cell culture media compositions and two values of scaffold rigidity. Differences in cell shape were achieved using interpenetrating polymer networks (IPNs). The mechanical and diffusional properties of the scaffolds and cell-matrix interactions were characterized. In addition, cell responses were evaluated in terms of cell spreading via gene and protein expression of differentiation markers. Cumulative results support, and extend upon previous work indicating that cell shape alone in 3D contexts does not significantly modulate hMSC differentiation, at least for the scaffold chemistry, range of modulus and culture conditions explored in this study. STATEMENT OF SIGNIFICANCE: In 2D contexts, inducing different cell shapes, by varying the curvature, area size and shape of a patterned surface, has significant effects on hMSC multipotency and the onset of cell differentiation. In contrast, in vitro studies in 3D contexts have suggested that hMSC differentiation does not directly correlate with cell shape. However, in 3D, the effects of cell morphology on hMSC differentiation have not yet been clearly established due to the chemical and physical properties being intertwined in 3D matrices. In this work, we studied the effects of round or elongated cell morphologies on the differentiation of hMSCs independently of scaffold composition, modulus, crosslink density and cell mediated matrix remodeling. Cumulative results support, and extend upon previous work indicating that cell shape alone in 3D contexts does not significantly modulate hMSCs differentiation commitment.

An improved correlation to predict molecular weight between crosslinks based on equilibrium degree of swelling of hydrogel networks.

Accurate characterization of hydrogel diffusional properties is of substantial importance for a range of biotechnological applications. The diffusional capacity of hydrogels has commonly been estimated using the average molecular weight between crosslinks (Mc ), which is calculated based on the equilibrium degree of swelling. However, the existing correlation linking Mc and equilibrium swelling fails to accurately reflect the diffusional properties of highly crosslinked hydrogel networks. Also, as demonstrated herein, the current model fails to accurately predict the diffusional properties of hydrogels when polymer concentration and molecular weight are varied simultaneously. To address these limitations, we evaluated the diffusional properties of 48 distinct hydrogel formulations using two different photoinitiator systems, employing molecular size exclusion as an alternative methodology to calculate average hydrogel mesh size. The resulting data were then utilized to develop a revised correlation between Mc and hydrogel equilibrium swelling that substantially reduces the limitations associated with the current correlation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1339-1348, 2018.

In vitro evaluation of a basic fibroblast growth factor-containing hydrogel toward vocal fold lamina propria scar treatment.

Scarring of the vocal fold lamina propria can lead to debilitating voice disorders that can significantly impair quality of life. The reduced pliability of the scar tissue-which diminishes proper vocal fold vibratory efficiency-results in part from abnormal extracellular matrix (ECM) deposition by vocal fold fibroblasts (VFF) that have taken on a fibrotic phenotype. To address this issue, bioactive materials containing cytokines and/or growth factors may provide a platform to transition fibrotic VFF within the scarred tissue toward an anti-fibrotic phenotype, thereby improving the quality of ECM within the scar tissue. However, for such an approach to be most effective, the acute host response resulting from biomaterial insertion/injection likely also needs to be considered. The goal of the present work was to evaluate the anti-fibrotic and anti-inflammatory capacity of an injectable hydrogel containing tethered basic fibroblast growth factor (bFGF) in the dual context of scar and biomaterial-induced acute inflammation. An in vitro co-culture system was utilized containing both activated, fibrotic VFF and activated, pro-inflammatory macrophages (MΦ) within a 3D poly(ethylene glycol) diacrylate (PEGDA) hydrogel containing tethered bFGF. Following 72 h of culture, alterations in VFF and macrophage phenotype were evaluated relative to mono-culture and co-culture controls. In our co-culture system, bFGF reduced the production of fibrotic markers collagen type I, α smooth muscle actin, and biglycan by activated VFF and promoted wound-healing/anti-inflammatory marker expression in activated MΦ. Cumulatively, these data indicate that bFGF-containing hydrogels warrant further investigation for the treatment of vocal fold lamina propria scar. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1258-1267, 2018.

Evaluation of the Osteoinductive Capacity of Polydopamine-Coated Poly(ε-caprolactone) Diacrylate Shape Memory Foams.

Recently, a novel shape memory polymer foam based on the photopolymerization of poly(ε-caprolactone) diacrylate (PCLDA) has been developed. These PCLDA foams enter a temporary softened state when briefly treated with warm saline (T saline > T m of PCLDA), allowing them to conform to irregular bone defect "boundaries" prior to shape setting. When coated with a mechanically stable polydopamine (PD) layer, these PCLDA foams have previously been demonstrated to induce hydroxyapatite deposition. In the present study, the osteoinductivity of these "self-fitting" PD-coated PCLDA (PD-PCLDA) materials was evaluated relative to uncoated PCLDA (U-PCLDA) controls using bone marrow-derived human mesenchymal stem cells (h-MSCs). When cultured in the absence of osteogenic media supplements, PD-PCLDA scaffolds expressed similar levels of Runx2, alkaline phosphatase, and osteopontin protein as U-PCLDA scaffolds cultured in the presence of osteogenic media supplements. In addition, PD-PCLDA scaffolds cultured without osteogenic supplements did not significantly promote undesired lineage progression (e.g., adipogenesis or chondrogenesis) of h-MSCs. Cumulatively, these data indicate that PD-PCLDA materials display increased osteoinductivity relative to U-PCLDA substrates. Future studies will examine tethered osteogenic factors or peptides toward augmenting the osteoinductive properties of the PD-PCLDA foams.

A bioactive "self-fitting" shape memory polymer scaffold with potential to treat cranio-maxillo facial bone defects.

While tissue engineering is a promising alternative for treating critical-sized cranio-maxillofacial bone defects, improvements in scaffold design are needed. In particular, scaffolds that can precisely match the irregular boundaries of bone defects as well as exhibit an interconnected pore morphology and bioactivity would enhance tissue regeneration. In this study, a shape memory polymer (SMP) scaffold was developed exhibiting an open porous structure and the capacity to conformally "self-fit" into irregular defects. The SMP scaffold was prepared via photocrosslinking of poly(ε-caprolactone) (PCL) diacrylate using a SCPL method, which included a fused salt template. A bioactive polydopamine coating was applied to coat the pore walls. Following exposure to warm saline at T>T(trans) (T(trans)=T(m) of PCL), the scaffold became malleable and could be pressed into an irregular model defect. Cooling caused the scaffold to lock in its temporary shape within the defect. The polydopamine coating did not alter the physical properties of the scaffold. However, polydopamine-coated scaffolds exhibited superior bioactivity (i.e. formation of hydroxyapatite in vitro), osteoblast adhesion, proliferation, osteogenic gene expression and extracellular matrix deposition.

Inorganic-organic hybrid scaffolds for osteochondral regeneration.

Ligament graft failure frequently results from poor integration of the replacement tissue with associated bone. Thus, the ability to regenerate the bone-ligament osteochondral interface would be advantageous in ligament reconstruction. At the osteochondral interface, the tissue transitions from a bone-like matrix to fibrocartilage. Therefore, a scaffold which promotes a spatially regulated transition in cell behavior from osteoblast-like to chondrocyte-like would be desirable. Previous research indicates that addition of inorganic components to organic scaffolds can enhance the deposition of bone-like matrix by associated osteoblasts. We therefore reasoned that a gradient in the inorganic content of a hybrid inorganic-organic scaffold may induce an osteochondral-like transition in cell phenotype and matrix production. To test this hypothesis, hydrogels were prepared from poly(ethylene glycol) (PEG) and star poly(dimethylsiloxane) (PDMS(star)). As anticipated, both the matrix deposition and phenotype of encapsulated osteoblasts varied with scaffold inorganic content, although the directionality of this modulation was contrary to expectation. Specifically, osteoblasts appeared to transdifferentiate into chondrocyte-like cells with increasing scaffold inorganic content, as indicated by increased chondroitin sulfate and collagen type II production and by upregulation of sox9, a transcription factor associated with chondrocytic differentiation. Furthermore, the deposition of bone-like matrix (collagen type I, calcium phosphate, and osteocalcin) decreased with increasing PDMS(star) content. The resistance of the PDMS(star)-PEG scaffolds to protein adsorption and/or the changes in gel modulus/mesh structure accompanying PDMS(star) incorporation may underlie the unexpected increase in chondrocytic phenotype with increasing inorganic content. Combined, the present results indicate that PDMS(star)-PEG hybrid gels may prove promising for osteochondral regeneration. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.