Flowable Polyethylene Glycol Hydrogels Support ... - ACS Publications

Subscriber access provided by YORK UNIV

Tissue Engineering and Regenerative Medicine

Flowable Polyethylene Glycol Hydrogels Support the in Vitro Survival and Proliferation of Dermal Progenitor Cells in a Mechanically-Dependant Manner Holly Sparks, Fraz Anjum, Queralt Vallmajo-Martin, Martin Ehrbar, Sepideh Abbasi, Michael Scott Kallos, and Jeff Biernaskie ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01294 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 22, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Flowable Polyethylene Glycol Hydrogels Support the in Vitro Survival and Proliferation of Dermal Progenitor Cells in a Mechanically-Dependent Manner Holly D Sparks1, †, Fraz Anjum2, 3, †, Queralt Vallmajo-Martin7, Martin Ehrbar7, Sepideh Abbasi1, Michael S Kallos2, 3, * and Jeff Biernaskie1, 4, 5, 6, * Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary 3330 Hospital Dr. NW Calgary, AB, T2N 4N1 Canada 2Pharmaceutical Production Research Facility, University of Calgary, 2500 University Dr. NW Calgary AB T2N 1N4 Canada 3Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, 2500 University Dr. NW., Calgary, AB, Canada, T2N 1N4 4Department of Surgery, Cumming School of Medicine 5Alberta Children’s Hospital Research Institute 6Hotchkiss Brain Institute, University of Calgary 3330 Hospital Dr. NW Calgary, AB, T2N 4N1 Canada 7 Department of Obstetrics, University Hospital Zurich, University of Zurich, Schmelzbergstr. 12, 8091 Zurich, Switzerland † Co-first authors: (Jeff Biernaskie: [email protected] and Michael Kallos: [email protected]) * Co-corresponding authors 1

Abstract Cell-based therapies have garnered considerable interest largely due to their potential utility for tissue regeneration in a variety of organs, including skin. Designing vehicles that enable optimal delivery and purposeful integration of donor cells within tissues will be critical for their success. Here, we investigate the utility of an injectable, self-polymerizing, fully synthetic hydrogel in supporting the survival, proliferation, and function of cultured adult dermal progenitor cells (DPCs) which may serve as a source of renewable cells to repair severe skin injuries or restore hair growth. We show that modifying the stiffness of these transglutaminase cross-linked poly (ethylene glycol) (TG-PEG) hydrogels significantly alters DPC behavior and phenotype; increasing stiffness promotes their differentiation and migration whereas softer gels maintained them in a proliferative state. We found that 2-3% TG-PEG was optimal to promote cell expansion and survival. Unexpectedly, DPCs grown in all conditions maintained their inductive function and thus generated de novo hair follicles. Our data suggests that TG-PEG hydrogels may be a versatile platform for stem and progenitor cell transplantation and fate specification whilst maintaining functional competence. Key words Hydrogel, Poly(ethylene glycol), skin derived precursors, wound healing, skin grafting, stem cells. 1. Introduction Wound healing following severe skin injury is limited by the disorganized and often exuberant response from dermal fibroblasts consequently leading to fibrotic scar. Scar tissue is highly dysfunctional and often leads to lifelong impairment for the patient 1. Thus, strategies designed to instead regenerate functional dermal tissue during wound healing is of great interest. To this end, recent work identified a specialized group of hair follicle dermal stem cells (hfDSCs) that function to continuously regenerate new mesenchymal cells within the hair follicle 2. When isolated and

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

expanded in vitro, these cells are coined dermal progenitor cells (DPCs). They have been shown to exhibit long-term self-renewal and not only regenerate neodermis after transplantation into rodent skin wounds, but also induce de novo formation of hair follicles 2-4. Similarly, DPCs (also referred to as SKPs, or skin-derived precursor cells in previous work) have been shown to possess multipotent potential through differentiation into adipogenic, osteogenic, and neuronal cells5. Importantly, to maintain this inductive function, DPCs must be cultured in suspension culture where single cells proliferate to form spherical aggregates. If instead cultured as an adherent monolayer on plastic, these cells lose their ability to regenerate new hair follicles and become reminiscent of other, non-regenerative dermal fibroblasts3, 6. As a result of these characteristics, interest in dermal progenitor cells as a renewable resource for dermal reconstruction has increased 3.

Before this potential can be realized, a suitable method of cell delivery must be identified to

ensure DPC viability and to provide the appropriate mechanical and biological cues that will enhance integration and tissue regeneration within a skin wound 7-8. The ideal substrate for skin wound healing and cell delivery has yet to be discovered. Practically, the substrate should be safe, biocompatible and easy to handle. When utilized for delivery of cellbased therapies, it should allow for sustained survival and proliferation while maintaining the cells’ endogenous function. Hydrogels in particular have shown great promise for skin wound applications as they share many characteristics with that of extracellular matrix, including that of their mechanical properties9. Importantly, three general categories of materials are available for the creation of these hydrogels: natural, synthetic, or a hybrid of the two. Natural polymers such as collagen, alginate, Matrigel, and chitosan have advantages of improved biomimetic and cell adhesion properties10. However, their limitations include potential immune response, rapid degradation, and variability between batches11-12. Synthetic materials such as PEG are much more reproducible and do not risk disease transmission, but typically have inferior properties for cell adhesion. As well, injectable hydrogel materials

13-14

have also been recently developed which greatly

simplify delivery and distribution of the substrate into abnormally shaped or tunneled wounds. Such technology could additionally be utilized for application beneath split thickness skin grafts (STSG; the current standard of care for large wounds), potentially supporting graft adhesion which could decrease the time of graft surgeries and improve graft take15. Importantly, such a system


Page 2 of 27

Page 3 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


currently is only available comprised of natural materials16. Here, we endeavored to develop a nonimmunogenic fully synthetic, injectable hydrogel, whose mechanical properties could be specifically tailored for a variety of clinical applications. Poly(ethylene glycol) (TG-PEG) hydrogels are cross-linked by factor XIIIa (FXIIIa), a transglutaminase involved in the coagulation cascade17. As this type of hydrogel is crosslinked under physiological conditions without the addition of chemical initiators, UV light, or extreme temperatures, cells can safely be incorporated before gelation and the gel remains injectable until it reaches body temperature. To overcome limitations of cell adhesion as is typically observed with synthetic polymers, we investigated the utility of cell-adhesion peptides (RGD) conjugated onto the polymers. Additionally, as the mechanical properties of substrates have been shown to greatly affect the function of cells18-19, we sought to investigate the phenotype and cell behavior of cells cultured across the range of mechanical properties compatible with 3D culture (1.5-3% TGPEG)20. Notably, we discovered that increasing hydrogel stiffness promotes differentiation and migration of DPCs, however growth in these 3D hydrogel environments preserves the functional ability of the cells to induce hair follicle formation. 2. Materials and Methods Materials FXIIIa substrate and cell adhesion peptides (immunograde, C18-purified, HPLC analysis: > 90%) were obtained from Bachem (Basel, Switzerland)

17.

PEG-vinyl sulfone (8-PEG-VS, mol wt.

40000) was purchased from NOF Europe (Grobbendonk, Belgium). Synthesis of PEG gel precursors Eight-arm PEG precursors containing FXIIIa substrate peptides were produced and characterized as described previously17. FXIIIa lysine donor substrate peptides containing a matrix metalloproteinase sensitive site (TG-MMPsensitive-Lys; Ac-FKGG-GPQGIWGQ-ERCG-NH2; mol. wt. 1717.9 g/mol), FXIIIa glutamine acceptor substrate peptides (TG-Gln; H-NQEQVSPL-ERCGNH2; mol wt. 1358.5 g/mol) and 8-PEG-VS were dissolved in spate vials using 0.3 M triethanolamine (pH 8.0). TG-MMPsensitive-Lys and TG-Gln peptides were reacted at 37°C for 2 h in separate vials to PEG-VS (1.2-fold molar excess of peptides over VS groups). The resulting products 8-PEG-Gln and 8-PEG-MMPsensitive-Lys were dialyzed (Snake Skin, MWCO 10K,



PIERCE, Rockford, IL, USA) for 3 days at 4°C against ultrapure water before they were lyophilized and analyzed by 1H NMR (Figure 1A,B).

Formation of PEG hydrogels Hydrogels were formed by FXIIIa-mediated crosslinking of 8-PEG-Gln and 8-PEG-MMP-Lys as previously described

17.

For this, FXIIIa (200 U/mL, CSL Behring, Switzerland) was activated

with thrombin (2 U/mL, Sigma–Aldrich, Switzerland) for 30 min at 37 °C and stored in small aliquots at -80 °C. Stoichiometrically balanced ([Lys]/[Gln] = 1) precursor solutions of n-PEGGln and n-PEG-MMP-sensitive-Lys were prepared in 50 mM Tris buffer pH 7.6 containing 50 mM CaCl2. Hydrogels with final dry mass contents 1 - 3% w/v were prepared by the addition of 10 U/mL thrombin activated FXIIIa at 37°C. Hydrogel discs were obtained by injecting the reaction mixture between two salinized microscopic glass slides (treated with SigmaCote, SigmaAldrich) separated by silicone spacers (~1mm). The crosslinking reaction was allowed to proceed for 1 h under humidified atmosphere at 37oC (Figure 1C). Swelling and Network Characterization Hydrogels were prepared according to the procedure described above and freeze dried. The initial dry mass (wd) of hydrogels was recorded before soaking in Tris buffer (50 mM, pH 7.6) for 24 h, changing the media three times in this period. The swollen mass (ws) of hydrogels was measured after 24 h. The mass swelling ratio (Qm) was calculated by using the formula ws/wd. The volumetric swelling (Qv) was calculated from the mass swelling ratio using the densities of hydrogel precursors and Tris buffer 21. 𝜌𝑝

𝑄𝑣 = 1 + 𝜌𝑠 (𝑄𝑚 ― 1) ---- (1) Where, p and s are densities of polymer and solvent used for gelation (water = 1.0 g/cm3). The density of the polymer blend was calculated by taking the percentage of the polymer in precursor solutions and using the density of PEG (1.12 g/cm3). The network mesh size (ξ) of hydrogels was evaluated using Qv according to modified Flory-Rehner theory 22. Rheology on swollen hydrogels Hydrogels were prepared as described above and equilibrated for 24 h in Tris buffer, pH 7.6 at 37°C. To determine hydrogel mechanical properties, storage modulus (Gʹ) and loss modulus (Gʹʹ)


Page 4 of 27

Page 5 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were measured at 25°C in a small strain oscillatory shear with an Anton Par Rheometer (Buchs/AG, Switzerland) in parallel plate geometry and a 20 mm diameter upper plate. The gap was adjusted starting from the original swollen hydrogel height and compressing the sample to reach a normal force of 0.15 N. Gap sizes for swollen hydrogels were generally between 1.0 and 1.5 mm. An amplitude sweep (Gʹ as a function of strain) was performed in order to confirm the linear viscoelastic regime and the measurements were conducted before the shear thinning phase. Measurements were conducted at a frequency of 0.05 to 10 Hz and constant 2% strain to obtain mechanical spectra. Rodent DPC isolation and culture All animal use was performed in accordance with the Animal Care Committee at the University of Calgary. DPCs were isolated from the back skin of P30 male GFP expressing Sprague Dawley rats (SLC, Japan) following an overdose of sodium pentobarbital (27.3mg/kg, i.p. injection), according to previously described protocols 4 . Primary dermal cells were expanded with bFGF (40ng/ml, BD Biosciences), PDGF-BB (25 ng/ml, R&D Systems), B27 supplement (2%, Invitrogen) and penicillin/streptomycin (1%, Invitrogen) in low glucose DMEM/F12 (3:1, Invitrogen). DPCs were plated at 30,0000 cells/ml in suspension culture flasks (Falcon) and grown in a humidified incubator at 37ºC and 5% CO2 in air. Media was refreshed every 3-4 days during expansion. Following primary aggregate (colony) formation, DPCs were dissociated to single cells using collagenase digestion (2mg/mL, Worthington) and re-plated at 30,000 cells/mL every 7 days. DPC encapsulation within hydrogels DPCs were expanded in suspension culture until a uniform population of spheroid colonies was achieved. For the encapsulation of single cells, collagenase was utilized to dissociate spheroid colonies then neutralized in media containing 10% Fetal Bovine Serum (FBS, ThermoFisher) and rinsed well to ensure no residual collagenase was present as this would rapidly degrade the hydrogel. For the encapsulation of spheroid colonies, these colonies were collected by centrifugation and rinsed as above before encapsulation. For cell encapsulation experiments 20ul hydrogels were formulated in presence of 50 µM cell adhesion ligand RGD-Gln (AcFKGGRDGSPG-NH2; mol wt. 1018.3 g/mol), unless indicated otherwise. DPCs were added to hydrogel precursor solutions either as single cells (3 x 104 cells/ 20µL gel) or as intact spheroid colonies (4-8 per 20µL gel, equivalent to 2-4.8 x 104 cells/ 20µL gel) prior to the addition of FXIIIa



and vortexing. Cell-laden hydrogels were allowed to polymerize for 1 hour at 37°C under humidified atmosphere and 5% CO2 before they were immersed in cell culture medium (Figure 1D).

Figure 1: Hydrogel creation, cell encapsulation, and experimental design. Structure of (A) 8arm PEG-Gln and (B) 8-arm PEG-Lys following the addition of RGD and (C) FXIIIa-mediated crosslinking of 8-PEG-Gln and 8-PEG-MMP-Lys in the presence of RGD. (D) Phase contrast image of rat DPC aggregates as grown in standard culture media. (E) GFP expression observed in all DPCs utilized in this experiment (F) Experimental design. DPCs were isolated and expanded from GFP+ rats. Resulting cell aggregates were either encapsulated whole or


Page 6 of 27

Page 7 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dissociated into single cells in 4 differing concentrations of gels. Then several assays were performed to assess cell functionality. Cell survival and proliferation For cell survival and proliferation studies, single cells were encapsulated in nine replicates per condition. On days 1, 3, and 7 of culture, cell-encapsulated hydrogels were incubated in Collagenase IV (5.0 mg/mL in PBS, Worthington) for 5-10 min and mechanically dissociated. Liberated cells were then collected in a 15ml conical tube containing 10% FBS in DMEM to neutralize collagenase, centrifuged for 6 min at 1200 RPM, and resuspended in PBS. Cells were then stained with trypan blue and live cells counted with a hemocytometer. Live cell numbers on days 3 and 7 were expressed relative to the number of cells present on day 1 to account for any inaccuracies in seeding. Stiffness Decay To determine their remodeling, hydrogels with different initial polymer content were formed in the absence of cells as well as in presence of single DPCs or DPCs aggregates. All hydrogels were kept under standard culture conditions (37°C under humidified atmosphere and 5% CO2). Rheology measurements were conducted at day 1, 3, 5 and 7 as described earlier in the method section.

Cell Migration After polymerization, cell-laden hydrogels were equilibrated in cell culture medium overnight then placed in separate wells of 12-well culture plates before 2.0 mL of complete medium was added to submerse the gels. Random positions were imaged inside the hydrogels using an inverse wide field microscope (DM IRBE; Leica, Wetzlar, Germany), equipped with a motorized stage and focus, and camera. Cell migration and spreading were followed up to 24 hours at 10 min intervals, by software-controlled image acquisition under high relative humidity at 37°C and 5 % CO2.



Immunofluorescence staining Single cell aggregates were cultured in standard conditions for 5 days prior to immunostaining. At this timepoint, gels were rinsed in PBS before cell fixation in situ in 4% Paraformaldehyde for 5 minutes under continuous agitation. Gels were rinsed again in PBS then blocked overnight at 4°C in 10% Donkey Serum/0.5% Triton-X PBS. Primary antibodies were diluted in 1% Donkey Serum in PBS and incubated overnight at 4°C. Antibodies used included Integrin alpha 8 (R&D AF4076), Integrin alpha 9 (R&D AF3827), and Pax1 (Sigma SAB2101727) at 1:500, Runx1-3 (Abcam ab92336) at 1:50, and Sox2 (Stemgent 09-0024) at 1:100. Secondary antibodies were used at 1:500 in PBS and incubated overnight at 4°C. Nuclei were labeled using Hoechst at 1:1000. Gels were then dissolved using collagenase and cells maintained in PBS for flow cytometry.

Flow cytometry Immunostained DPCs isolated from each gel condition were assessed with flow cytometry to determine the fraction of cells expressing each marker of interest. All events (2,500-3,000) were collected for each marker of interest and gates were set according to unstained and secondary only controls (in the absence of isotype controls). Cytometry data presentation was done with FlowJo (Treestar).

In vivo hair follicle formation assay Hair follicle formation assays were performed as previously described 2. Briefly, DPCs were grown in hydrogels for 7 days and then retrieved from gels and dissociated into single cells by collagenase digestion. Simultaneously, epithelial aggregates were collected from P0 C57BL/6J


Page 8 of 27

Page 9 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Jax # 000664) pups following enzymatic digestion of the epidermis. Cell numbers were standardized across all conditions such that 100,000 live DPCs were combined with 5,000 epithelial aggregates in 80μl DMEM for each injection. Six-week old male nude mice (Nu/Nu) were anaesthetized and the cell suspension representing each condition was injected subcutaneously in 4 separate locations on the dorsal back. After 2 weeks, mice were euthanized by CO2 and the back skin collected. Skin was fixed in 2% PFA overnight, rinsed in PBS, then the subcutaneously tissues gently dissected to reveal the injection sites. Imaging was performed with fluorescence stereomicroscopy (Zeiss Lumar V12 Stereo). Newly formed hair follicles were confirmed to contain GFP+ mesenchymal cells in the dermal papilla. These follicles were then counted manually by a blinded observer.

Statistical Analysis Data were entered into statistical software (GraphPad Prism) and significance set at p < 0.05. For comparisons between multiple groups, a one-way ANOVA was performed with Tukey’s multiple comparisons test utilized to detect differences between groups. All graphs are representative of mean ± SEM.

Results: Flowable PEG Hydrogels Polymerize at body-temperature (37°C) PEG hydrogels with concentrations ranging from 1.5% to 3.0% were created and mechanically characterized (Figure 1). All concentrations of PEG hydrogel precursors combined with FXIIIa. were found to be stable for up to 2 days as a flowable hydrogel when kept cold. Once the substrate was exposed to body temperature (37C), crosslinking was activated, and polymerization was



Page 10 of 27

initiated. The polymerization time depended on both the concentration of hydrogel precursor as well as FXIIIa (Figure 2A). Specific ranges of FXIII studied were selected based upon the maximum amount needed to fully crosslink the material, thus making crosslinking not dependent upon naturally encountered FXIII as would be expected in a wound environment. Specific hydrogel mechanical characteristics identified 24 hours following encapsulation are summarized below. Table 1: Swelling and Network Characterization of Acellular Hydrogels. PEG Swelling (Q) Storage modulus (Pa) Mesh size (ξ) (nm)

1.5% 42 ± 3.2 92 ± 19

2.0% 38 ± 5.1 249 ± 12

2.5% 31± 4.4 360 ± 14

3.0% 23 ± 2.8 494 ± 11

37.5 ± 0.3

34.8 ± 0.2

32.3 ± 0.5

27.0 ± 1.1

PEG Hydrogels Stability is Concentration - Dependent To determine their in vitro remodeling, PEG hydrogels with concentrations ranging from 1.5% to 3.0% were created both in the absence and in the presence of single cells or cellular aggregates. Mechanical characterization showed that empty PEG hydrogels maintain their mechanical properties over a minimum of 7 days under cell culture conditions (Figure 2B). In contrast, hydrogels that contained DPCs either as single dispersed cells (Figure 2C) or as spheroids (Figure 2D) softened in a PEG concentration dependent manner (Figure 2C). Interestingly, the presence of single cells resulted in a more pronounced softening of the hydrogels as compared to the presence of spheroids (Figure 2E). This is most obvious for the 1.5% hydrogel, which in presence of single dispersed cells completely degraded after 5 days of culture. Overall, the degradation of PEG hydrogels was diminished when cells were embedded as aggregates rather than as single cells (Figure 2C and D), as well as when lower cell densities were employed (Figure 2F).


Page 11 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2: Mechanical Characteristics of Hydrogels. (A) Polymerization time (seconds) of 1.5% TG-PEG by varying concentrations of FXIII. Storage modulus of (B) hydrogels alone, (C) hydrogels containing single-cells, and (D) hydrogels containing aggregates. (E) Percent mechanical decay of acellular and cellular gels at day 7. (E) Degradation time for gels containing 30,000-90,000 cells per hydrogel at day 7. * Significant difference (p

Flowable Polyethylene Glycol Hydrogels Support ... - ACS Publications

Recommend Documents