Collagen/kerateine multi-protein hydrogels as a thermally stable extracellular matrix for 3D <i>in vitro</i> models

Zuniga, Kameel; Gadde, Manasa; Scheftel, Jacob; Senecal, Kris; Cressman, Erik N.K.; Dyke, Mark Van; Rylander, Marissa Nichole

doi:10.1080/02656736.2021.1930202

Cited by 13 publications

(14 citation statements)

References 110 publications

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“…Although keratin is not present in the dermal layer, we wanted to determine whether keratin promotes the differentiation of keratinocytes when it is included in the ECM upon which the keratinocytes are seeded. Because keratin is naturally found in the epidermis, we employed a 50/50 w%/w% collagen/keratin hydrogel, which allowed the spreading and proliferation of NHDFs, as previously shown by the Rylander laboratory [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

“…The 50/50 w%/w% C/KTN hydrogels were prepared by combining stock collagen and KTN dissolved in neutralizing buffer of equal volume. Previously in our laboratory, we tested both 50/50 and 30/70 C/KTN hydrogels, and observed low proliferation and growth of fibroblasts in 30/70 C/KTN hydrogels, while cell growth was observed for 50/50 C/KTN hydrogels, comparable to 100% collagen hydrogels [ 36 ]. Therefore, we only tested 50/50 C/KTN against 100% collagen hydrogels.…”

Section: Methodsmentioning

confidence: 99%

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Keratin Promotes Differentiation of Keratinocytes Seeded on Collagen/Keratin Hydrogels

et al. 2022

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Keratinocytes undergo a complex process of differentiation to form the stratified stratum corneum layer of the skin. In most biomimetic skin models, a 3D hydrogel fabricated out of collagen type I is used to mimic human skin. However, native skin also contains keratin, which makes up 90% of the epidermis and is produced by the keratinocytes present. We hypothesized that the addition of keratin (KTN) in our collagen hydrogel may aid in the process of keratinocyte differentiation compared to a pure collagen hydrogel. Keratinocytes were seeded on top of a 100% collagen or 50/50 C/KTN hydrogel cultured in either calcium-free (Ca-free) or calcium+ (Ca+) media. Our study demonstrates that the addition of keratin and calcium in the media increased lysosomal activity by measuring the glucocerebrosidase (GBA) activity and lysosomal distribution length, an indication of greater keratinocyte differentiation. We also found that the presence of KTN in the hydrogel also increased the expression of involucrin, a differentiation marker, compared to a pure collagen hydrogel. We demonstrate that a combination (i.e., containing both collagen and kerateine or “C/KTN”) hydrogel was able to increase keratinocyte differentiation compared to a pure collagen hydrogel, and the addition of calcium further increased the differentiation of keratinocytes. This multi-protein hydrogel shows promise in future models or treatments to increase keratinocyte differentiation into the stratum corneum.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Keratin Promotes Differentiation of Keratinocytes Seeded on Collagen/Keratin Hydrogels

et al. 2022

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“…Keratins have high thermal stability, excellent cytocompatibility, and can be obtained from readily available sources [14,28,29]; thus, they have been used in numerous published studies as scaffolds for regenerative medicine [5-7, 16, 17]. The few prior reports of 3D printing with keratin-based bio-inks have relied on the addition of photosensitive compounds or composite mixtures of keratins with other materials to achieve the desired properties for successful 3D printing [2,[18][19][20][21][22]30].…”

Section: Discussionmentioning

confidence: 99%

Extrusion 3D printing of keratin protein hydrogels free of exogenous chemical agents

Brodin¹,

Prentice²,

Neff³

et al. 2022

Biomed. Mater.

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Keratins are a class of intermediate filament proteins that can be obtained from numerous sources including human hair. Materials fabricated from keratins offer desirable characteristics as scaffolds for tissue engineering, including intrinsic cell adhesion sequences and tunable degradation kinetics. The capacity to create 3D printed constructs from keratin-based bio-inks generates unique opportunities for spatial control of scaffold physicochemical properties to direct scaffold functions in ways not readily achieved through other means. The aim of this study was to leverage the controllable rheological properties of keratin hydrogels to create a strategy for extrusion 3D printing of keratin bio-inks without the use of exogenous rheological modifiers, crosslinking agents, or photocurable resins. The rheological properties of keratin hydrogels were tuned by varying two parameters: (a) the ratio of keratose (obtained by oxidative extraction of keratin) to kerateine (obtained by reductive extraction of keratin); and (b) the weight percentage of total keratin protein in the gel. A computational model of the dispensing nozzle for a commercially available extrusion 3D printer was developed to calculate the needed pneumatic printing pressures based on the known rheological properties of the gels. Keratin hydrogel constructs, of varying keratose/kerateine ratios and total keratin weight percentages, were 3D printed in cylindrical geometries via extrusion 3D printing. Rheology and degradation studies showed that gels with greater relative kerateine content exhibited greater flow resistance and slower degradation kinetics when submerged in phosphate buffered saline solution at 37 °C, owing to the presence of cysteine residues in kerateine and the capability of forming disulfide bonds. Total keratin weight percentage was found to influence gel yield stress, with possible implications for tuning filament fidelity. Findings from this work support the use of keratose/kerateine ratio and total keratin weight percentage as handles for modulating rheological characteristics of keratin hydrogels to enhance printability and control scaffold properties.

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“…[9,14,16,[45][46][47][48] Hydrogels have been widely implemented to develop tissue engineered constructs that mimic various aspects of the tumor microenvironment. [5,[19][20][21][22][23][24][25][26][27][28][29] For instance, natural hydrogels have been implemented to model the tumor microenvironment using proteins such as collagen, gelatin, and fibrin and polysaccharides such as hyaluronic acid and alginate have been incorporated to produce semi-synthetic hydrogels. [49][50][51] While these hydrogels provide relevant biochemical properties, batch to batch variability, viscosity, and limitations concerning independent control of mechanical and biochemical properties are often challenging obstacles to overcome with when using these materials.…”

Section: Discussionmentioning

confidence: 99%

“…It is well established that cell-ECM interactions play a key role in governing cancer cell fate. [5,[19][20][21][22][23][24][25][26][27][28][29] In this context, we previously developed sixteen unique hydrogel formulations composed of three components: (1) proteolytically degradable PEG (PEG-PQ), (2) integrin ligating PEG-RGDS, and (3) a nondegradable crosslinker NVP. By systematically tuning the concentration of PEG-RGDS and NVP, hydrogel adhesivity and degradability were varied, inducing different phenotypes in the parental triple negative breast cancer line (MDA-MB-231) and three of its organotropic sublines (BRM2a-831s, LM2-4175s, BoM-1833s).…”

Section: Hydrogel Characterizationmentioning

confidence: 99%

Tuning Hydrogel Adhesivity and Degradability to Model the Influence of Premetastatic Niche Matrix Properties on Breast Cancer Dormancy and Reactivation

Reyes

Slater

2022

Advanced Biology

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Dormant, disseminated tumor cells (DTCs) can persist for decades in secondary tissues before being reactivated to form tumors. The properties of the premetastatic niche can influence the DTC phenotype. To better understand how matrix properties of premetastatic niches influence DTC behavior, three hydrogel formulations are implemented to model a permissive niche and two nonpermissive niches. Poly(ethylene glycol) (PEG)‐based hydrogels with varying adhesivity ([RGDS]) and degradability ([N‐vinyl pyrrolidinone]) are implemented to mimic a permissive niche with high adhesivity and degradability and two nonpermissive niches, one with moderate adhesivity and degradability and one with no adhesivity and high degradability. The influence of matrix properties on estrogen receptor positive (ER+) breast cancer cells (MCF7s) is determined via a multimetric analysis. MCF7s cultured in the permissive niche adopted a growth state, while those in the nonpermissive niche with reduced adhesivity and degradability underwent tumor mass dormancy. Complete removal of adhesivity while maintaining high degradability induced single cell dormancy. The ability to mimic reactivation of dormant cells through a dynamic increase in [RGDS] is also demonstrated. This platform provides the capability of inducing growth, dormancy, and reactivation of ER+ breast cancer and can be useful in understanding how premetastatic niche properties influence cancer cell fate.

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Collagen/kerateine multi-protein hydrogels as a thermally stable extracellular matrix for 3D in vitro models

Abstract: Rylander (2021) Collagen/kerateine multi-protein hydrogels as a thermally stable extracellular matrix for 3D in vitro models,

Cited by 13 publications

References 110 publications

Keratin Promotes Differentiation of Keratinocytes Seeded on Collagen/Keratin Hydrogels

Keratin Promotes Differentiation of Keratinocytes Seeded on Collagen/Keratin Hydrogels

Extrusion 3D printing of keratin protein hydrogels free of exogenous chemical agents

Tuning Hydrogel Adhesivity and Degradability to Model the Influence of Premetastatic Niche Matrix Properties on Breast Cancer Dormancy and Reactivation

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