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. 2022 Oct;110(10):2323-2337.
doi: 10.1002/jbm.b.35080. Epub 2022 May 9.

Native human collagen type I provides a viable physiologically relevant alternative to xenogeneic sources for tissue engineering applications: A comparative in vitro and in vivo study

Tânia Baltazar  1 , Nilabh S Kajave  2 , Marco Rodriguez  3 , Srija Chakraborty  3 , Bo Jiang  4   5 , Aleksander Skardal  3   6 , Vipuil Kishore  2 , Jordan S Pober  1 , Mohammad Z Albanna  7   8
Affiliations

Native human collagen type I provides a viable physiologically relevant alternative to xenogeneic sources for tissue engineering applications: A comparative in vitro and in vivo study

Tânia Baltazar et al. J Biomed Mater Res B Appl Biomater. 2022 Oct.

Abstract

Xenogeneic sources of collagen type I remain a common choice for regenerative medicine applications due to ease of availability. Human and animal sources have some similarities, but small variations in amino acid composition can influence the physical properties of collagen, cellular response, and tissue remodeling. The goal of this work is to compare human collagen type I-based hydrogels versus animal-derived collagen type I-based hydrogels, generated from commercially available products, for their physico-chemical properties and for tissue engineering and regenerative medicine applications. Specifically, we evaluated whether the native human skin type I collagen could be used in the three most common research applications of this protein: as a substrate for attachment and proliferation of conventional 2D cell culture; as a source of matrix for a 3D cell culture; and as a source of matrix for tissue engineering. Results showed that species and tissue specific variations of collagen sources significantly impact the physical, chemical, and biological properties of collagen hydrogels including gelation kinetics, swelling ratio, collagen fiber morphology, compressive modulus, stability, and metabolic activity of hMSCs. Tumor constructs formulated with human skin collagen showed a differential response to chemotherapy agents compared to rat tail collagen. Human skin collagen performed comparably to rat tail collagen and enabled assembly of perfused human vessels in vivo. Despite differences in collagen manufacturing methods and supplied forms, the results suggest that commercially available human collagen can be used in lieu of xenogeneic sources to create functional scaffolds, but not all sources of human collagen behave similarly. These factors must be considered in the development of 3D tissues for drug screening and regenerative medicine applications.

Keywords: 3D construct; cancer modeling; collagen type I; hydrogel; xeno.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mohammad Z. Albanna is a founding member of Humabiologics, a start-up developing human-derived biomaterials for tissue engineering, which provided the HumaDerm products tested in this study. Mohammad Z. Albanna had no role in data collection. The other authors declare that they have no competing interests.

Figures

FIGURE 1
FIGURE 1
Variation in tissue source, species, and isolation technique has a significant impact on the physical properties of collagen type I (A) Gelation kinetics, (B) polymerization rate determined by linear regression,(C) swelling ratio, (D) in-vitro collagenase degradation with time, (E) representative stress-strain curves, and (F) compressive modulus of different collagen type I hydrogels. (* indicates p < .05, ** indicates p < .01, *** indicates p < .001, **** indicates p < .0001).
FIGURE 2
FIGURE 2
SEM imaging of hydrogels shows differences in collagen fiber morphology due to variation in tissue source, species, and extraction methods. LIVE/DEAD imaging indicates that hMSCs remain viable over time on collagen type I hydrogels. AlamarBlue assay revealed enhanced cell metabolic activity on rat tail and human skin derived collagen type I hydrogels. (A) SEM and Live/Dead assay to assess cell viability. Scale bar: 5 μm - SEM images; 200 μm - Live/Dead staining images. (B) Quantification of cell viability. (C) Quantification of cell number per surface area. (D) Quantification of cell metabolic activity on collagen type I hydrogels using alamarBlue assay–(* indicates p < .05 when comparing with bovine collagen at the same time point, # indicates p < .05 when comparing with human fibroblasts collagen at the same time point, & indicates p < .05 when comparing with human placenta collagen at the same time point).
FIGURE 3
FIGURE 3
ATP quantification demonstrates that matrix-supported colorectal 3D tumor constructs largely respond in a dose dependent manner to chemotherapy agents Regorafenib and 5-fluorouracil. ATP luminescence readings of colorectal cancer Caco-2 3D constructs on days 1, 4, and 7 following drug administration at concentrations of 1, 10, and 100 μm (n=4). Conditions include Regorafenib- (Top) and 5- fluorouracil- (Bottom) treated rat tail collagen type I and human skin collagen type I 3D tumor constructs. Statistical significance: †, * and # - p < .05 between different drug concentrations at a given timepoint; ‡ - p < .05 between timepoints at a given concentration.
FIGURE 4
FIGURE 4
LIVE/DEAD visualization of colorectal cancer 3D constructs qualitatively corroborates quantitative data indicating that 3D tumor constructs largely respond in a dose dependent manner to chemotherapy agents. Fluorescent microscope images of colorectal cancer Caco-2 3D constructs stained with calcein AM (green) and ethidium homodimer-1 (red) at days 1, 4, and 7 following drug administration at concentrations of 1, 10, and 100 μm (n = 3). Conditions include Regorafenib- (Top) and 5-fluorouracil- (Bottom) treated rat tail collagen type I and human skin collagen type I 3D tumor constructs. Green–viable cells stained with calcein AM; Red–dead cells stained with ethidium homodimer-1. Scale bar: 100 μm.
FIGURE 5
FIGURE 5
Effects of rat tail collagen type I and human skin-derived collagen type I containing hydrogels on human vessel formation in vivo. (A) Photographs of hydrogels prior and 14 days post-implantation into the abdominal wall of a SCID/bg mouse model. Scale bar: 5 mm. (B) Assessment of gel contraction by measurements of hydrogel weight at the time of implantation and after explant. Data are shown with standard error of mean (SEM) (n = 3; p < .05). (C) Representative H&E images of hydrogels 14 days post-implantation, showing that rat tail and human skin-derived collagen type I containing hydrogels support vessel formation in vivo. Immunohistochemical analysis of F4/80 expression shows different degrees of infiltration of murine macrophages. Inset images represent low magnification views of hydrogel crosssections. Scale bar: 500 μm. (D) Quantification of area of F4/80+ staining shows that degree of infiltration is particularly higher in hydrogels containing human skin-derived collagen type I (* indicates p < .05). (E) Hydrogels formulated with rat tail and human-skin derived collagen type I contain vascular structures 14 days post-implantation. Presence of mouse microvessels and basement membrane deposition was assessed by staining with mouse CD31 and human collagen IV antibodies, respectively. To demonstrate that human EC-lined vessels were perfused, fluorescein ulex was injected 30 min before explant. Staining shows that several human vessels have anastomosed with mouse vessels and became perfused in vivo in rat tail and human skin-derived collagen type I hydrogels. Post-harvest staining with rhodamine ulex shows a significant higher number of human EC-lined vessels in the center of these hydrogels that were not perfused at the time of fluorescein ulex infusion. Scale bar: 500 μm (high magnification images: 100 μm). (F) Quantification of area of infused ulex shows no significant difference in the number of perfused human EClined vessels between conditions. (ns indicates p > .05).
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References

    1. Shafiee A, Atala A. Tissue engineering: toward a new era of medicine. Annu Rev Med. 2017;68:102715. doi:10.1146/annurev-med-102715-092331 - DOI - PubMed
    1. León-López A, Morales-Peñaloza A, Martínez-Juárez VM, Vargas-Torres A, Zeugolis DI, Aguirre-Álvarez G. Hydrolyzed collagen-sources and applications. Molecules. 2019;24:4031. doi:10.3390/molecules24224031 - DOI - PMC - PubMed
    1. Silvipriya KS, Kumar KK, Bhat AR, et al. Collagen: animal sources and biomedical application. J Appl Pharm Sci. 2015;5:123–127.
    1. Angele P, Abke J, Kujat R, et al. Influence of different collagen species on physico-chemical properties of crosslinked collagen matrices. Biomaterials. 2004;25:2831–2841. doi:10.1016/j.biomaterials.2003.09.066 - DOI - PubMed
    1. Lin YK, Liu DC. Comparison of physical–chemical properties of type I collagen from different species. Food Chem. 2006;99:244–251. doi:10.1016/j.foodchem.2005.06.053 - DOI

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