Promoting Adipogenesis Using a Collagen VI–Heparin Sulfate

Research Note pubs.acs.org/IECR

Promoting Adipogenesis Using a Collagen VI−Heparin Sulfate Coating: Applications in Tissue Engineering for Wound Healing Laura T. Beringer,† Shaohua Li,† Ethan J. Kallick,‡ Kelly J. Shields,§ Erin M. Faight,§ Francis Cartieri,‡ Ariel Aballay,∇ Howard Edington,‡ and Saadyah Averick*,† †

Neuroscience Disruptive Research Laboratory, Allegheny Health Network Research Institute, Pittsburgh, Pennsylvania 15212, United States ‡ Department of Surgery, Allegheny Health Network, Pittsburgh, Pennsylvania 15212, United States § Lupus Center of Excellence, Autoimmunity Institute, Department of Medicine, Allegheny Health Network, Pittsburgh, Pennsylvania 15212, United States ∇ West Penn Burn Center, Allegheny Health Network, Pittsburgh, Pennsylvania 15212, United States S Supporting Information *

ABSTRACT: Treating burn-related injuries remains challenging, because of donor site morbidity, bacterial infection, and major scarring. Recent advances have led to the development of promising natural and synthetic polymer scaffolds, the objective of which is to regenerate skin using a “topdown” approach, in which the top layers of the skin populated by fibroblasts and keratinocytes are regenerated first. An often-overlooked component of the integumentary system is the subcutaneous adipose tissue, which has been implicated in a variety of activities, including wound healing. We have created a novel coating composed of collagen VI (col VI) and heparin sulfate (HS) that promotes adipocyte attachment and adipogenesis at a rate significantly faster at both early and late time points (5 and 23 days), compared to a noncoated control. In addition, Vicryl, which is a biodegradable surgical implant mesh, was coated with the optimized col VI−HS ratio. Coated Vicryl had an increased number of attached adipocytes with more-pronounced growth, when compared to uncoated Vicryl. These findings suggest that the combination of optimized col VI−HS coating and the fibrous woven Vicryl may have excellent potential in developing skin graft applications using a paradigm-shifting “bottom-up” approach.

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endocrine organ and has been implicated in metabolism regulation, hormone secretion, and even inflammatory responses.16 During the proliferative phase of healing, Schmidt and Horsley14 have shown that adipocytes are recruited to the wound bed and concurrently aid in regeneration, along with fibroblasts. In addition, Nakajima et al. have shown a strong link between adipocytes and extracellular matrix (ECM) deposition for regeneration in the dermal layer of the skin.15 Adipocytes secrete both heparin sulfate and collagen VI during maturation, thereby maintaining their phenotype, and have shown enhanced growth on collagen-VI-coated substrates.10,11 Because of the probable key role that adipocytes play within the complex wound healing cascade, they are a prime source of innovative research in the field of soft tissue engineering. In order to better understand adipocyte biology, with regard to their growth and maturation, we sought inspiration from the components of the adipocyte ECM. We have identified and

egeneration of soft tissue after injury, especially after a severe burn, remains a prominent challenge in regenerative medicine.1,2 When skin integrity is compromised because of thermal, chemical, or electrical trauma, a complex signaling cascade involving cytokines, biomolecules, and recruitment of fibroblasts and keratinocytes initiates the wound healing process.3,4 Most often, a moderate to severe burn is accompanied by life-threatening morbidities, including systemic and local infections, fluid imbalance, and disturbance of body homeostasis.12 Various allograft, autograft, or xenograft scaffolds have been developed to initiate wound healing by promoting cell migration and proliferation to improve both the graft and patient survival.5−9 However, these types of surgeries are plagued with difficulties, such as a lack of donor tissue and the high probability of infection; ultimately, these issues necessitate solutions that can be implemented through developments in the field of tissue engineering.12,13 Interestingly, the base layer of the dermis includes subcutaneous adipose tissue and is often overlooked in the development of skin grafts. Intradermal adipocytes have recently been implicated in the complex wound healing cascade. Adipose tissue is now considered a major © 2016 American Chemical Society

Received: Revised: Accepted: Published: 12687

September 15, 2016 November 16, 2016 November 18, 2016 November 18, 2016 DOI: 10.1021/acs.iecr.6b03567 Ind. Eng. Chem. Res. 2016, 55, 12687−12692

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Industrial & Engineering Chemistry Research

Figure 1. Immunofluorescent and bright-field micrographs of adipocytes grown on control TCP and col VI-HS coatings after 5 days: (a) control, (b) 100 col VI, (c) 80/20 col VI-HS, (d) 60/40 col VI-HS, (e) 40/60 col VI-HS, (f) 20/80 col VI-HS, and (g) 100 HS. Panel (h) shows the AdipoRed fluorescence index, normalized to control. Scale bars = 100 μm; inset shows a bright-field micrograph (width = 100 μm). Red arrows indicate lipid cluster development.

tion, and growth, it should dramatically increase the adhesion of mature adipocytes and dramatically increase the rate of preadipocyte differentiation into mature adipocytes, when applied to biodegradable tissue scaffolding. Herein, we detail the coating optimization and validation of activity.

optimized a novel combination of heparin sulfate and collagen VI (col VI-HS) that dramatically enhances adipocyte attachment as well as differentiation of preadipocytes into adipocytes. Col VI-HS was used to coat a Vicryl mesh to simulate a possible burn wound healing scaffold. Vicryl is an FDAapproved bioabsorbable polyglycolic acid fibrous mesh that is used in a variety of temporary organ and wound support surgical repairs, but is not a typical scaffold material for adipocyte tissue engineering. We chose to explore its use in the context of adipocytes, because of its known biocompatibility and the potential to provide a suitable framework to regenerate adipocyte tissue. Our coating is easy to formulate and, given its functionality in promoting adipocyte differentiation, matura-

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RESULTS AND DISCUSSION

Culture of Adipocytes on Tissue Culture Polystyrene. Adipocytes were grown in 24-well tissue culture plates coated with the novel col VI-HS ratios to determine the effect that these ECM proteins have on adipocyte maturation. Adipogenesis was measured through the use of a hydrophobic fluorescent dye that interacts with the lipid droplets within 12688

DOI: 10.1021/acs.iecr.6b03567 Ind. Eng. Chem. Res. 2016, 55, 12687−12692

Research Note

Industrial & Engineering Chemistry Research

Figure 2. Merged immunofluorescent and bright-field micrographs of adipocytes grown on TCP and col VI-HS coatings after 23 days: (a) control, (b) 100 col VI, (c) 80/20 col VI-HS, (d) 60/40 col VI-HS, (e) 40/60 col VI-HS, (f) 20/80 col VI-HS, and (g) 100 HS. Panel (h) shows the AdipoRed fluorescence, normalized to control. All scale bars = 100 μm; inset shows a bright-field micrograph (width = 100 μm).

maturing adipocytes. A higher fluorescent signal indicates a larger quantity of droplets. Optimal VI-HS ratio was taken to be that which had the highest index of normalized lipid content at 5 and 23 days. The FDA-approved woven fibrous mesh Vicryl was then prepared with the optimal coating ratio of col VI-HS, as determined through the first series of experiments, in order to assess if the coating could enhance adipocyte attachment. Adipocytes began to differentiate within 48 h after exposure to adipocyte differentiation media (ADM), demonstrated by observable formation of lipid droplets through a bright-field microscope. After 5 days of growth, adipocytes were assayed with AdipoRed; immunofluorescent and bright-field microscopy images were then taken to quantify the amount of lipid droplet formation, which is indicative of adipogenesis. As shown in Figure 1, marked differences among the control TCP well and the col VI-HS are present. Lipid droplets are more numerous and appear larger on every coating combination of col VI and HS, as demonstrated by the increased amount of orange/red fluorescence in the immunofluorescent micrographs and the red arrows highlighting lipid clusters in the bright-field images (Figures 1b−g). Adipocytes have begun to take on a rounded morphology, which is in stark contrast with the fibroblast-like morphology of the human subcutaneous preadipocytes (HPAD) prior to differentiation (S1). A quantitative measurement of AdipoRed fluorescence was also made, as shown in Figure 1h. Adipocytes grown on all coatings

of col VI and HS demonstrated a significantly larger amount of red fluorescent signal (p < 0.01), compared to the control adipocytes. The 20/80 col VI-HS coating had the highest fluorescent signal, which was calculated to be more than 200% of the control adipocytes. The pure col VI and the 80:20 col VIHS had the lowest fluorescent signals, compared to the rest of the coatings, but these were still significantly higher than that of the control cells. After 23 days of growth, adipocytes adopted a rounded morphology, much like a native white fat cell, and contain multiple lipid droplets that have increased in size and in number, as shown in Figure 2. This morphology change is apparent compared to Figure 1 at day 5. Interestingly, adipocytes grown on the pure HS coating retained a more triangular shape (Figure 2g), whereas the other coatings with decreased concentrations of HS promoted the round morphology (Figures 2b−f). The quantitative fluorescent measurement revealed an interesting discovery at this time point for adipocytes grown on control TCP or the col VI-HS coatings. As shown in Figure 2h, only the 20:80 col VI-HS coating had a significant increase in red fluorescent signal (p < 0.05), compared to control. The other coatings had either significantly less red signal (p < 0.01) or no significant difference, compared to the control. These data suggest that the promotion of adipogenesis and lipid droplet formation on col VI-HS coatings is time-dependent, with regard to the adipocyte 12689

DOI: 10.1021/acs.iecr.6b03567 Ind. Eng. Chem. Res. 2016, 55, 12687−12692

Research Note

Industrial & Engineering Chemistry Research

Figure 3. MicroCT images of unseeded Vicryl meshes: (a) uncoated Vicryl and (b) coated 20:80 col VI-HS Vicryl. (c) Attenuated total reflectance− Fourier transform infrared (ATR-FTIR) spectra of each mesh. (d) Table of mesh characteristics, as determined by microCT.

amount of HS present in the coating. In addition, morphological characteristics of the uncoated and coated Vicryl mesh were assessed using microCT post-scan analysis. Individual threads are readily observed in the uncoated mesh, having a much larger surface area, albeit with a smaller volume per thread, when compared to the coated mesh. The coating covers the individual fibers resulting in a decrease of continuity; however, the underlying pattern of the threads is discernible and the distance between the coated threads is just slightly greater, when compared to the uncoated mesh (Figure 3d). Growth of Adipocytes AdipoMesh. Based on the success with col VI-HS coatings in tissue culture wells, it was hypothesized that adipocytes preferentially grow on coated Vicryl as well. Utilizing the AdipoRed stain for the cells and the natural blue autofluorescence of the mesh, Figure 4 reveals the morphology after 9 days of growth. Figure 4a displays adipocytes grown on the uncoated Vicryl mesh, which did support cell growth, as expected, because Vicryl is a suitable material for soft organ support in surgery. However, Figure 4b reveals a significant amount of cells grown on the Vicryl mesh coated with 20:80 col VI-HS. The adipocytes are visible and appear to have a mature morphology, similar to what was observed with the coating experiments. In contrast, Figure 4d reveals a prominent increase in the amount of cells present on the coated Vicryl mesh. These differences are better highlighted in Figures 4c and 4d with immunofluorescent micrographs taken at 40× magnification. A larger number of cells can be seen growing on each fiber of the coated mesh versus the control mesh, indicating that the col VI-HS coating improved adipocyte attachment. Primary Adipocyte Adhesion to AdipoMesh. In order to further explore the cellular attachment properties of the coating and mesh combination, mouse primary adipocytes were freshly isolated and placed onto coated and uncoated Vicryl samples and allowed to incubate for 5 min. After 5 min, media was washed off and replaced for imaging to determine if cells would attach better to a coated matrix. Mouse adipocytes are very round and possess a range of size distribution. Interestingly, the coated Vicryl allowed a larger number of cells remaining after the wash, suggesting that adipocytes prefer to adhere to the col VI-HS coating. To quantify the human adipocyte results, ImageJ was utilized to threshold the images and count the region of interest (ROI) sites that appeared bright red (red fluorescent protein- RFP) on each type of mesh. Figure 4g (S2)

growth cycle. After 3 weeks of culture, the col VI-HS-treated adipocytes contain similar amounts of lipids, compared to control, in stark contrast to the five-day time point where significant differences were observed (Figure 1). In addition, the fluorescent intensity shift for adipocytes grown on control and the optimized coating from day 5 to day 23 is apparent, as adipocytes retain a much larger signal at both time points when grown on the 20:80 col VI-HS coating, compared to the control. This shift is unlikely to be caused by the hydrophobicity of the col VI-HS coating itself, as the fluorescent dye is estimated to have a similar excitation peak with and without the coating (see Magenau et al.,17 2015 for the estimation method). Because of its performance in this series of experiments, the 20:80 ratio was used in the subsequent Vicryl coating experiments. Culture of Adipocytes on Matrix Coated Vicryl (AdipoMesh). Characterization of AdipoMesh. Adipocytes were grown on both coated and uncoated Vicryl mesh in order to explore the possibility of this material as a scaffold for subcutaneous fat tissue engineering. Because Vicryl is an FDAapproved material, it would be much easier to test in a clinical capacity, if shown to be promising. In addition, Vicryl has not been researched as a scaffolding material for adipocytes, but it is widely used for a variety of surgical applications and possesses favorable biocompatibility with a variety of soft tissue and thus may have good material properties for adipocyte growth and maturation. Prior to adipocyte seeding, however, material characterization and imaging with microCT was done on both the coated and uncoated Vicryl meshes in order to reveal the scaffold morphology and verify that the coating protocol was successful. As shown in Figure 3, both meshes display a woven fibrous appearance; however, the uncoated Vicryl (Figure 3a) has a much smoother and more uniform fibrous appearance, compared to the coated Vicryl (Figure 3b). In addition, bright regions on the surface of the fibers can be seen in Figure 3b, likely due to the col VI-HS coating. Figure 3c verified coating deposition through attenuated total reflectance−Fourier transform infrared (ATR-FTIR) analysis, as coated Vicryl meshes reveal new peaks characteristic of proteins (col VI) and sulfates (HS). There is a large and pronounced peak at ∼1620 cm−1 in the coated Vicryl mesh, which is most likely an amide I band from the col VI. A peak shift accompanied by new peak formation is observed at ∼1030 cm−1, which is indicative of sulfonated groups, which would be consistent with the large 12690

DOI: 10.1021/acs.iecr.6b03567 Ind. Eng. Chem. Res. 2016, 55, 12687−12692

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Industrial & Engineering Chemistry Research

Figure 4. Immunofluorescent micrographs of human adipocytes on Vicryl mesh: (a) low-magnification uncoated Vicryl mesh human adipocytes, day 9 (scale bar = 300 μm); (b) low-magnification coated Vicryl mesh human adipocytes; day 9 (scale bar = 300 μm); (c) medium-magnification uncoated Vicryl mesh human adipocytes, day 9 (scale bar = 400 μm); (d) medium-magnification coated Vicryl mesh human adipocytes, day 9 (scale bar = 400 μm); (e) high-magnification image of uncoated Vicryl mesh fibers (scale bar = 100 μm); (f) high-magnification image of adipocytes clustering on coated Vicryl fibers (scale bar = 100 μm). Panel (g) shows an estimation of relative adipocyte cell number labeled as the red fluorescent protein (RFP) regions of interest (ROIs) on meshes.

culture of adipocytes, as measured after 5 and 23 days, compared to other ratios of colVI:HS and uncoated tissue culture plates. The matrix was coated onto Vicryl mesh as a potential dressing to promote burn wound healing. Adipocytes had statistically significant growth on the coated versus bare Vicryl. The matrix-coated Vicryl was utilized to isolate primary mouse adipocyte from solution, because of the rapid adhesion of these cells onto the scaffold. This report will be followed by detailed in vitro and in vivo assessment of the 20:80 col VI:HS coating in adipocyte characterization and burn wound healing animal models.

reveals the significant (p < 0.01) difference in cellular attachment of adipocytes on coated versus uncoated Vicryl mesh.

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CONCLUSIONS We have identified a biological coating that appears to enhance adipocyte attachment and maturation. Adipocytes have been recently implicated as important regulators in the soft tissue wound healing cascade, and these materials hold promise for innovative materials-based research to treat burn wounds. Using the natural matrix of adipocytes as inspiration, we identified a 20:80 col VI:HS as a superior coating for the tissue 12691

DOI: 10.1021/acs.iecr.6b03567 Ind. Eng. Chem. Res. 2016, 55, 12687−12692

Research Note

Industrial & Engineering Chemistry Research

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(14) Schmidt, B. A.; Horsley, V. Intradermal adipocytes mediate fibroblast recruitment during skin wound healing. Development 2013, 140 (7), 1517−1527. (15) Nakajima, I.; Muroya, S.; Tanabe, R.; Chikuni, K. Extracellular matrix development during differentiation into adipocytes with a unique increase in type V and VI collagen. Biol. Cell 2002, 94 (3), 197−203. (16) Coelho, M.; Oliveira, T.; Fernandes, R. Biochemistry of adipose tissue: An endocrine organ. Arch. Med. Sci. 2013, 9 (2), 191−200. (17) Magenau, A. J. D.; Saurabh, S.; Andreko, S. K.; Telmer, C. A.; Schmidt, B. F.; Waggoner, A. S.; Bruchez, M. P. Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation. Biomaterials 2015, 66, 1−8.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.6b03567. Materials and methods, as well as supplementary figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 412-359-4943. E-mail: [email protected]. ORCID

Saadyah Averick: 0000-0003-4775-2317 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Allegheny Health Network Research Institute for funding this project. This contribution was identified by Joram Slager (SurModics, Inc.) as the Best Presentation in the session “Polymer Applications & Characterization in Medical Devices Industry” of the 2016 ACS Spring National Meeting in San Diego, CA.

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REFERENCES

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DOI: 10.1021/acs.iecr.6b03567 Ind. Eng. Chem. Res. 2016, 55, 12687−12692