3D Biomaterial Matrix to Support Long Term, Full Thickness, Immunocompetent Human Skin Equivalents with Nervous System Components

Abstract

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (~1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery.

1. Introduction

The human skin is complex both in terms of structure and in physiology, but there has been limited success in developing complex tissue engineered skin equivalents that can function for extended time frames in vitro (e.g., weeks and months), and that simultaneously include nerve/immune components and the hypodermis (subcutis).

Current human skin equivalents (HSEs) are used clinically for many applications, including trauma, burns, chronic wounds and surgeries[1–3]. Separately, in vitro HSEs are mainly used in research and development as alternatives to animal testing for applications such as pharmaceutical development, in vitro diagnostics (toxicology, irritation, etc.), cosmetics testing, and wound healing models. State of the art clinical HSEs typically only contain 1–2 cell types (keratinocytes and fibroblasts) in a thin collagen sheet. Current commercially available replacements such as Apligraf, Dermagraft, and Orcel have successfully been used for the treatment of burns, skin grafts/wound covers, and chronic wounds. Commercially available in vitro systems like EpiSkin and Epiderm are typically used for the analysis of drug delivery/permeability, sensitization, and wound healing [4,5]. Non-standard research systems have an advantage of tunability, and thus can be more complex, and many tissue models include important cell types of the skin including melanocytes [6], endothelial cells [7,8], neurons [9–11], and immune cells [12]. However, to our knowledge there is no current HSE model that includes a hypodermis/immune component with a nerve component, which are known to be important for many complex skin functions and homeostasis [13,14].

The contraction of the collagen matrix (the most common HSE biomaterial for in vitro use) with time further limits HSE tissue utility, usually resulting in relatively short term in vitro studies [15]. This contracting matrix also limits the HSE tissue physiological relevance, as more robust systems necessitate matrices that can support extended experimental duration, mechanical handling and also sequential operations to combine different layers to form full thickness systems. Commercially available systems lack nerves and macrophages which are all located in the dermis [5,16]. Further, most models do not contain adipocytes of the hypodermis, which are increasingly thought to be important for skin regeneration following wounding, and historically known to be necessary as an energy source as well as a shock-absorber of the skin [17–19]. While many 3-dimensional (3D) cell and tissue models for skin exist, they also lack other vital components such as blood vessels, nerves, and glands [16]. Therefore, these HSEs are incomplete without the neuro-immuno-cutaneous system which have implications in skin homeostasis and function [20]. To our knowledge, no existing HSE contain both adipocytes and neurons.

Skin explants contain numerous cell types, maintain the extracellular matrix and the 3D structure of the tissue, and can be relatively simple to obtain. However, issues including patient variability, sourcing, and donor health are of concern [16,17]. Explants have been explored for re-innervation, via rat DRGs cultured underneath a dermal explant, which demonstrated nerve fibers could grow through the dermis and epidermis after 10 days [20]. Explant models present certain benefits through the maintenance of the 3D structure and functions of the skin, and have shown promise in in vitro studies for pharmaceuticals [16,17]. However, like the co-culture models, the nervous and vascular system are not functional in these explants, and there are significant issues with sample variability, accessibility, and a decrease in viability associated with these tissues [16,17].

Recent investigations into the role of both the nervous system and the hypodermis in skin models have been explored, in order to enhance the understanding of paracrine signaling within the skin and to improve the relevance of current models [10,11,14–16]. For example, both human keratinocytes and human fibroblasts were shown to individually recruit porcine DRGs from a central compartment [11]. This work exemplifies an important function of skin cells; however, it was carried out in a 2-dimensional environment without immune response considered. Thus, we propose to improve physiological relevance, first by identifying a suitable biomaterial matrix to support longer term in vitro cultivation, and then adding function with immune (through adipose tissue resident macrophages) and neuronal components.

This study focuses on increasing tissue complexity and stability for long term skin cultures, for analysis of the neuro-immuno-cutaneous network and its implications on inflammation. This was accomplished by first generating a new composite protein biomaterial matrix based on a silk-collagen system, to overcome the longstanding limitations with matrix contraction, and thus provide a new supporting matrix for complex skin tissue layers and cells. Silk has been successfully utilized as a biomaterial for several skin-based systems and wound healing models [15,21–23]. We previously reported a full-thickness skin model which included a hypodermis composed of a silk-based adipose layer seeded with adipose stem cells with a collagen HSE cultured on top [15]. However, this initial tissue model did not include this new composite matrix reported here, or the innervation, immune cells, and endothelial cells, and was not used for long-term analysis. Therefore, an improved tissue model with lipoaspirate-seeded hypodermis tissue was used in the current study to contain the relevant hypodermal cell types: adipocytes, pre-adipocytes, endothelial cells, smooth muscle pericytes, and inherent immune cells [24]. In addition, we incorporated human induced neural stem cells (hiNSCs) into our HSE constructs, which are generated through the direct reprogramming of dermal fibroblasts, are robust and maintain neural phenotype even in complex co-cultures [25]. These hiNSC lines are generated by the direct reprogramming of human somatic cells, without the requirement of a pluripotent intermediate. The hiNSCs differentiate into neurons in less than one week independent of media composition, and the cells maintain their neuronal phenotype even in non-neurogenic microenvironments, making them ideal for the type of complex innervated co-culture tissue model described herein.

These full thickness, innervated, silk-collagen-based HSEs are an advance upon the state of the art, commercially available HSEs which typically are composed of only 1 layer and 1–2 cell types [5,26]. This in vitro tissue model to account for the neuro-immuno-cutaneous network of the skin could be highly relevant in applications ranging from inflammation/injury to drug delivery, with particular focus on chronic or long term studies. The system can also be tuned to investigate patient-to-patient effects, and ultimately may be genetically engineered to develop highly specific, human disease models—without relying on animal or explant tissue sources.

2. Methods

2.1 Materials

Cell culture media, supplements, and materials were purchased from ThermoFisher Scientific unless otherwise noted. Transwells, horseradish peroxidase (HRP) (Type IV), and hydrogen peroxide were purchased from Sigma (St. Louis, MO). Bovine collagen (3mg/mL) was purchased from Advanced Biomatrix (San Diego, CA). Antibodies were purchased from Abcam (Anti-beta III Tubulin, CD68, Alexafluor secondaries) (Cambridge, MA). DAPI was purchased from Sigma.

2.2 Silk processing

Standard silk processing protocols were followed as described with either a 60-minute or 30-minute extraction time [27]. 60-minute extracted silk solution was utilized to generate the artificial dermis (instructions in supplement). To form silk scaffolds, lyophilized 30-minute extracted silk was reconstituted in 17% (w/v) hexafluoro-2-propanol (HFIP). Salt was sieved at 500–600 μm pore size, and 2 mL of silk was poured over 6.8 grams of sieved salt in a polyethylene container for 2 days and sealed. The remaining HFIP could evaporate by opening the seal for 1 day in a fume hood, after which the containers were submerged in methanol for 1 day followed by an additional day of evaporation in a fume hood. Then the vials were rinsed in 2 L beaker of deionized water, changing the water up to 6 times over a 3-day period. The silk scaffolds were then carefully cut into cylinders (2 mm height × 20 mm diameter) and autoclaved before use. Up to 1 day prior to use, scaffolds were submerged in maintenance media.

2.3 Cell maintenance

Primary neonatal foreskin fibroblasts (Lonza, Portsmouth, NH) were maintained in fibroblast media (S1). Primary neonatal keratinocytes (Lonza, Portsmouth, NH) were maintained in KGM Gold supplemented with KGM Bullet kits (Lonza Portsmouth, NH). Human induced neural stem cells (hiNSCs) were maintained as previously described [25]. All cells were maintained in a 37°C/5% CO₂ incubator.

2.4 Adipose isolation and scaffold preparation for hypodermis

Primary human adipose tissue was obtained from abdominoplasty procedures with institutional review board approval (Protocol #0906007) at the Lahey clinic (Burlington, MA, USA). Briefly, adipose tissue was dissected with blunt dissection tools to isolate adipose tissue. The adipose tissue was liquefied in a blender. The pre-warmed scaffolds were submerged in the liquefied adipose tissue in a 50 mL conical tube and placed in the incubator for 30 minutes. After 30 minutes the scaffolds were individually placed into 6 well plates and kept in the incubator for another 2 hours until maintenance media was added (Table S1).

2.5 Preparation of immuno-competent HSEs with nervous system components

2.5.1 Dermis and epidermis

Figure (1), Table (1) and supplementary tables (S1–S4) contain details on composition, media components and timing [15]. First, all relevant components from Table S4 were combined (i.e. 10× EMEM, Glutamax, Fetal Bovine Serum, and Sodium bicarbonate). For collagen-only gels, collagen (on ice) was added to the solution, carefully mixed, and then fibroblasts were added immediately prior to transfer of the gel solution onto 12-well insert transwells (Sigma). For silk-collagen gels, silk and collagen (both on ice) were added to the solution, carefully mixed, HRP was added and mixed, fibroblasts were then added and carefully mixed. Finally, hydrogen peroxide was added to the solution, quickly mixed, and immediately the gel solution was transferred onto transwells. Primary fibroblast-containing collagen or silk-collagen gels were prepared 14 days prior to the start of an experiment and maintained in maintenance media for 1 week. Primary keratinocytes (3×10⁵ cells/μL) in EPI1 media (Table S2) were seeded directly onto the fibroblast-containing collagen or silk-collagen gels after removal of media and a 20-minute waiting period to slightly dry the gels. After seeding, the plates were kept in the biosafety hood for 15 additional minutes to ensure keratinocyte attachment. A total of 250 μL of EPI1 media was added to the surface of the gels over the following 24 hours in 50 μL increments; with approximately 3 mL of EPI1 added to the bottom of the transwells at time of seeding. Dermal gels were raised to air-liquid interface one week after seeding with keratinocytes, and maintained at air-liquid interface throughout the study duration. Resultant silk-collagen gels were ~2.2 mm thick by D42; collagen gels were ~0.5 mm by D42.

Figure 1.

Experimental timeline for the formation of the full thickness, immuno-competent human skin equivalent with nervous system components.

The development of the HSE started by seeding the hypodermis (a porous silk sponge soaked with lipoaspirate from patient-donated abdominoplasty) (−21D). The next step was to create the dermis, a silk-collagen gel seeded with dermal fibroblasts (−14D), which was then coated in keratinocytes (−7D). At the start date (D0), the hypodermis was coated with a hiNSC-containing collagen gel, and placed underneath the dermal gel, and interfaced throughout culture up to 42 days (D42). Histological hematoxylin and eosin staining (right) of epidermis and hypodermis layers (scales = 100 μm). D= day; ALI = Air-liquid interface; hiNSCs = human induced neural stem cells.

Table 1.

Experimental nomenclature and descriptions.

A
Sample Group	Cells added	Description
Skin explant	None	Patient biopsy
Hypodermis	None	Silk-sponge with lipoaspirate
Skin	Keratinocytes, fibroblasts	Silk-collagen gel
Skin + Hypodermis	Keratinocytes, fibroblasts	Silk collagen gel (dermis), silk sponge with lipoaspirate (hypodermis)
Skin + Hypodermis + Nerve	Keratinocytes, fibroblasts, hiNSCs	Silk collagen gel (dermis), silk sponge with lipoaspirate (hypodermis) and hiNSCs

B
Sample Group	Description
Skin explant	Patient biopsy
Hypodermis	Silk-sponge with lipoaspirate
Acellular C	Collagen gel
Dermis C	Collagen gel with fibroblasts
Acellular SC	Silk-collagen gel
Dermis SC	Silk-collagen gel with fibroblasts
Dermis SC + Hypodermis	Silk-collagen gel with fibroblasts interfaced with hypodermis below

(A) Experimental groups and content description. (B) Experimental groups for mechanical behavior analysis, on differences between silk-collagen and collagen-only gels.

2.5.2 Hypodermal coating and interface with dermis

New transwells were used for the addition of the hiNSC-containing gels. The hiNSCs were enzymatically trypsinized, dissociated and labeled using lipophilic fluorescent dye Vybrant^™ DiD (Invitrogen) according to manufacturer’s protocol to monitor their growth in the HSEs. Briefly, hiNSCs were added to a collagen gel seeded directly onto transwell membranes. Then, single lipoaspirate scaffolds (the hypodermis) were placed on top of the hiNSC gel (one per well) and placed back in the incubator for 10 more minutes. Carefully, the hypodermis scaffold was then flipped so that both sides were coated in the hiNSC collagen gel, and placed in the incubator again for an additional 15 minutes. Once the gels were almost formed, the dermal component was placed on top of the hiNSC-coated hypodermis which remained interfaced throughout culture. Lastly approximately 3 mL of cornification media was added to the bottom of the transwell. The complete HSE was subjected to media changes 3 times a week for 6 weeks (Table S3).

2.6 Cytokine array

Cell culture media were frozen in aliquots at −20°C until analysis. Samples were analyzed with a Human cytokine antibody array (Abcam ab133996) per product instructions and imaged via a chemiluminescent imager (Syngene). Cytokines are defined in the supplementary information (Table S7). For analysis, each array was normalized to its own positive control, and samples were presented as a percentage of the positive control intensity. Each bar represents the average of 2 normalized cytokine spot replicates.

2.7 Durometer

A Rex gauge 1600 series Type OO (McMaster Carr, Elmhurst, IL) was used to analyze durometer readings for each HSE. The durometer was held vertically on samples measured via the gravity of the durometer impact on the sample; the durometer was kept in place for 20 seconds per measurement and the reading was recorded (all equilibrated to room temperature).[28] Five measurements were made per sample in different locations.

2.8 Mechanical compression analysis

Samples were removed from culture after 6 weeks and cut with an 8-mm biopsy. Unconstrained compression was performed using a TA Instruments RSA3 Dynamic Mechanical Analyzer (TA Instruments, New Castle, DE). Samples were analyzed via a percentage of height with respect to strain up to 35% of the initial height for each individual sample to account for size disparity between samples and groups. At least five measurements were performed per sample type, with error representing mean ± standard error of the mean (SEM).

2.9 Immunohistochemistry (IHC)

HSEs were fixed immediately after being taken out of culture using 10% neutral buffered formalin for 1 hour. Samples were then washed 3 times with PBS (20 minutes per wash) with one final overnight PBS rinse. Then, samples were submerged in 30% (w/v) sucrose solution in PBS overnight before embedding with Tissue-Plus O.C.T. Compound. Cryosections were cut at −20°C (stage and chamber temperature) using a cryostat (Leica) to a thickness of 50 μm and placed onto SuperFrost slides. Briefly, samples were washed twice with PBS (5 minutes per wash) prior to blocking with 10% goat serum (ThermoFisher Scientific, Waltham, MA) for 1 hour. Primary antibodies were then incubated for 1–4 hours at room temperature followed by a PBS rinse (10 minutes, twice). Secondary antibodies were incubated for 4 hours at room temperature or overnight at 4°C followed by a PBS rinse (10 minutes, twice). Samples were mounted using BrightMount Plus (Abcam) and protected from light until analysis. Images were taken with a Leica TCS SP8 confocal microscope with a 20× air objective.

2.10 Histology

Standard hematoxylin and eosin staining procedures were used on 50 μm frozen sections. Histological samples were mounted using Permount. Images were taken with a Keyence All-in-One Fluorescent Microscope (BZ-X710) with a 20× air objective.

2.11 Liquid chromatography tandem mass spectroscopy (LC-MS/MS)

Excised gel bands were cut into approximately 1 mm³ pieces. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure [29]. Gel pieces were washed and dehydrated with acetonitrile for 10 minutes followed by removal of acetonitrile. Pieces were then completely dried in a speed-vac. Rehydration of the gel pieces was with 50 mM ammonium bicarbonate solution containing 12.5 ng/μl modified sequencing-grade trypsin (Promega, Madison, WI) at 4°C. After 45 minutes, the excess trypsin solution was removed and replaced with 50 mM ammonium bicarbonate solution to just cover the gel pieces. Samples were then placed in a 37°C room overnight. Peptides were later extracted by removing the ammonium bicarbonate solution, followed by one wash with a solution containing 50% acetonitrile and 1% formic acid. The extracts were then dried in a speed-vacuum (~1 hr). The samples were then stored at 4°C until analysis.

On the day of analysis, the samples were reconstituted in 5 – 10 μl of HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nano-scale reverse-phase HPLC capillary column was created by packing 2.6 μm C18 spherical silica beads into a fused silica capillary (100 μm inner diameter × ~30 cm length) with a flame-drawn tip [30]. After equilibrating the column each sample was loaded via a Famos auto sampler (LC Packings, San Francisco CA) onto the column. A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid).

As peptides eluted they were subjected to electrospray ionization and then entered into an LTQ Orbitrap Velos Pro ion-trap mass spectrometer (ThermoFisher Scientific). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program, Sequest (ThermoFisher Scientific) [31]. All databases include a reversed version of all the sequences and the data was filtered to between a one and two percent peptide false discovery rate.

2.12 Quantitative real time polymerase chain reaction (qRT-PCR)

Samples were stored in RNAlater^™ (ThermoFisher Scientific) at −80°C until use according to manufacturer protocol. Total RNA was isolated using the RNeasy Mini kit (Qiagen, Germantown, MD), and cDNA was generated using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer protocol. Quantitative RT-PCR was performed on the iQ5 Real-Time PCR Detection System (BioRad, Cambridge, MA) using the QuantiTect SYBR Green PCR Kit (Qiagen) and normalized against the housekeeping gene 18S. All primers were purchased from ThermoFisher Scientific (S8).

2.13 Statistics

Data was analyzed from sample groups from 2 main experiments, using cells/tissue from 2 patient groups (Patient A, Patient B). Assays were performed in triplicate, presented as average from at least 2 independent experiments with error bar ± SEM. Data analysis was performed using OriginPro (version 8.6). One-way ANOVA with a Tukey post hoc test was performed to determine statistical significance, where P-values < 0.05 were considered significant.

3. Results

3.1 Biomaterials design

State of the art HSEs are typically composed of only collagen, which is subject to contraction and deterioration with time (Figure 3A). Compared to the same volume and cell number of a collagen gel, silk-collagen gels retain 87% of their initial diameter after 6 weeks in culture (where collagen retained only 56%). In material evaluation tests, acellular collagen gels lost approximately 3.14 times more of its initial mass in 72 hours compared to silk-collagen gels (Figure S1). The improved material properties of silk-collagen were likely due to interaction of the available tyrosine residues on silk and collagen; this suggested that the resultant hydrogel was less susceptible to degradation, physical contraction, and was therefore more conducive to long-term biomaterials testing than collagen only gels. This silk-collagen system as a dermal material for HSEs also maintained its thickness with time. Dermal cells were spatially distributed through the dermis, an epidermal layer spanned the entire sample, and the porous silk-based hypodermis was coated with hiNSC-containing collagen gel interfacing neurons with the adipose tissue (Figure 1).

Figure 3.

Immunohistochemistry of HSEs for determination of neural and immune cells.

(A) Skin + Hypodermis + Nerve groups at 6 weeks (C) of culture demonstrated neural cells in the hypodermis of the model apparent in overlapping red (DiD-hiNSCs) and green (TUJ1) channel. CD68 signal (yellow) was apparent throughout the duration of culture, indicating the presence of macrophages clustered in the hypodermis region. Groups containing hypodermis (A, B) had a higher CD68 signal than Skin only (C). (B) The Hypodermis group had CD68 signal, but did not have DiD-hiNSC or TUJ1 positive staining, indicating that the Hypodermis did not contribute any neural cells. (B, C samples were imaged after 3 weeks in culture). All samples (A, B, C) are average projections of 20–30 μm; (A, B) were taken in hypodermis-sections, (C) is a cross-section of a dermal HSE with the epidermis exposed in the top left. White #’s signify areas with high amounts of hiNSCs (red and green channels) and white &’s signify regions with high amounts of immune staining (yellow channel). (Scales = 100 μm, blue = DAPI, red = Vybrant^™ DiD-dyed hiNSCs, green = TUJ1, yellow = CD68).

3.2 Mechanical analysis of HSEs

The experimental groups for the mechanical studies are defined in Table 1B, analysis in Figure 2.

Figure 2.

Mechanical behavior of silk-collagen gels.

(A) Diameter change with time demonstrated that silk-collagen dermal gels maintain 87.5% of their initial diameter, whereas collagen-only gels contracted to 56.3% of the initial diameter by 42 days (6 weeks) in culture (□silk-collagen, △ collagen-only, note: materials change color in different media conditions). (B) Durometer (Type OO) measurements after 6 weeks in culture demonstrated that acellular and cellular collagen gels had higher durometer values corresponding to hardness. Both cellular and acellular silk-collagen gels were closer to the value of skin explant (measured as received). All groups were significantly different from one another at P < 0.001, except for the dermis (collagen) group compared to acellular (collagen), which were not statistically significant. (C, D) Samples were analyzed in unconfined compression after 6 weeks in culture with respect to layer (hypodermis, dermis, and full thickness dermis + hypodermis) in comparison to a skin explant (measured as received). The hypodermis alone had a higher stiffness than other groups, but when interfaced with the silk-collagen dermis the stiffness was reduced to a value similar to the skin explant. Error bars are ± SEM.

3.2.1 Durometer

Collagen gels (both cellular and acellular) had significantly higher durometer readings than silk-collagen gels (both cellular and acellular) and an explant (from abdominoplasty) (Figure 2B). While the silk-collagen materials also had significantly higher durometer readings than the explant, the both silkcollagen materials were more similar to human skin tissue than the collagen materials.

3.2.2 Unconfined compression

There were no significant differences between the Young’s Modulus or stiffness of the different layers and combinations of the HSE materials, but there were trends observed in the stiffness of each material (Figure 2C). The Hypodermis alone was almost twice as stiff as the Dermis, Dermis + Hypodermis layers combined, or the Skin explant.

3.3 Evaluation of immune cells present within HSEs

To verify the presence of immune cells in the HSE system qRT-PCR was performed using CD68 and CSF1 (macrophage markers) on cultured hypodermis sample at 1, 3, and 6-week post-surgery. With respect to week 1, CD68 at week 3 had a 12-fold increase, and by week 6 a 16-fold increase; CSF1 at week 3 had a 38.32-fold increase and by week 6 had a 170-fold increase. Pro-inflammatory markers IL-6 and RANTES had increased fold changes from week 1 while NOS2 (pro-inflammatory) and adipose marker ACRP30 decreased (Figure S2, Table S6).

IHC on frozen tissue sections using marker CD68 verified that samples with hypodermis had a high signal, regardless of HSE group (Figure 3A–E). The staining pattern for CD68 indicates either macrophage clusters or possibly free CD68 protein released during the adipose tissue processing. This distinct CD68 positive staining was not apparent in the Skin control group (Figure 3F) where no adipose tissue was added.

3.4 Evaluation of nerves within HSEs with time

To visualize the hiNSCs with time, hiNSCs were pre-dyed before addition to the HSEs (D-1 of experiment, Figure 1) with Vybrant^™ DiD cell dye to distinguish between any tissue resident neurons originating in the lipoaspirate, and by IHC of β-III Tubulin (TUJ1) on frozen sections. The TUJ1 and Vybrant^™ DiD tracer overlapped (green and red channels, Figure 3A–C) suggesting that the hiNSCs remained in the hypodermis up to 6 weeks and can form dense neural networks.

3.5 Quantification of immune response and HSE function

In general, there was less overall pro-inflammatory cytokine secretion from the Skin-only groups—which only contained keratinocytes and fibroblasts (Figure 4A). Cytokine definitions can be found in Table S7. Skin-only groups demonstrated time-dependent decreases in MCP-1, IL-6, IL-8, and GRO secretion. Between hypodermis-only groups, Patient A and Patient B exhibited opposite trends with Patient A activity decreasing with time while Patient B increases (more notably in IL-6, IL-8, and MCP-1). Adding the hypodermis to the skin (Skin + Hypodermis group), cytokine activity remains high, and elevated over the Skin group. The addition of nerve, (Skin + Hypodermis + Nerve) seems to elicit patient-dependent responses, where the absolute value changes (Patient A higher than Patient B), but the trends are similar, with activity highest at D1 then decreasing through D42; whereas without the nerve (in Skin + Hypodermis) activity peaks at D7 then decreases. MCP-1 is noted in all groups at D1 and D7 but not at later time-points. IL-6 levels were quantified in Figure 4C. In general, the hypodermis has the highest secretion levels, increasing with time; however the Skin, Skin + Hypodermis, and Skin + Hypodermis + Nerve groups have their highest signal at week 1 which decreases by week 6. Macrophage colony stimulating factor, MCSF-1, had a low signal from the Skin-only group but in the groups with a hypodermis component the signal was higher (Figure 4D). In the skin groups containing hypodermis, the MCSF-1 signal was lower than the hypodermis alone, which may be related to the lower IL-6 levels detected in these samples compared to the hypodermis alone group.

Figure 4.

Addition of the hypodermis to HSEs increased release of pro-inflammatory cytokines, with dynamic time and patient specific changes.

(A) Pro-inflammatory cytokine array with respect to group, and patient-source (for lipoaspirate used in hypodermis) demonstrated that groups with the hypodermis had higher levels of cytokines than the group without (skin alone). (B) Glycerol secretion with respect to group demonstrated that hypodermis alone had initially higher values but was depleted by 6 weeks (42 days) of culture, whereas Skin + Hypodermis and Skin + Hypodermis + Nerve group had lower but sustained glycerol release with time. (C) A similar trend was noted with interleukin-6 (IL-6) secretion with time, where hypodermis-containing groups were generally higher than Skin, and the addition of skin (to Skin + Hypodermis and Skin + Hypodermis + Nerve) lowered the IL-6 values. (D) Macrophage colony-stimulating factor (MCSF1) secretion was higher in the Hypodermis than in the hypodermal-containing groups, and lowest in Skin, suggesting that the more complex models may be immune-competent due to the addition of the Hypodermis. Error bars are ± SEM, data represents averaged results from Patient A and Patient B experiments.

Glycerol secretion was monitored to assess adipose function. At 6 weeks, there was a sharp decline in glycerol secretion in the Hypodermis from previous weeks. In the Skin + Hypodermis + Nerve group the secretion was lower, but stable over time (Figure 4B).

3.6 Proteomic analysis via LC-MS/MS

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is equipped to identify specific proteins of interest via improved protein database quality and availability [32]. Overall, between Patient A and B there was a factor of 2.68 fewer proteins identified in Patient B (Figure 5). However, the top identified protein between all sample types (± hypodermis, nerves) and Patient groups (A or B) transferrin (TF). Other highly detected proteins include vinculin (VCL), lacto-transferrin (LTF), complement 3 (C3), and alpha-2-microglobulin (A2M). Of all the groups, the samples with nerves have the most detected proteins attributed to the complexity of the model (Skin + Hypodermis + Nerve: 165, 60; Skin: 85, 41; Skin + Hypodermis: 135, 40 (Patient A, Patient B unique detected proteins, respectively)).

Figure 5.

Proteomic analysis via LC-MS/MS with respect to patient and sample group.

LC-MS/MS detection of proteins between Patient and sample group, presented in heat maps of all the proteins detected, and as the LC top 10 detected proteins within each sample group with time (bar graphs). Patient A had 303 total detected unique proteins between all sample groups, whereas Patient B had only 113 unique proteins. However, of the top 10 proteins, between Patient there were similarities—TF (transferrin)—was high in all samples, and in general many of the proteins in the top 10 are associated with inflammation, cell adhesion, motility, and extracellular matrix formation.

4. Discussion

Since degradation of explants and contraction of collagen limit current HSE models to relatively short time frames of functional utility, an appropriate biomaterial must be designed that preserves the physiological properties of the skin’s collagen matrix while maintaining its structure for long-term culture. Silk derived from Bombyx mori silkworms has been used in numerous tissue engineering applications due to its cyto- and bio-compatibility, resistance to degradation, and versatility [24,27,33]. In particular, tyrosine residues in silk fibroin can form di-tyrosine bonds via a HRP – hydrogen peroxide reaction which does not form toxic byproducts, resulting in a mechanically tunable, biocompatible hydrogels suitable for a variety of tissue matrices [33]. Collagen type 1 has about 8 tyrosine residues per 3,000 amino acids and a triple helical structure which can degrade due to proteolytic activity [34,35]. Without modification, collagen rapidly contracts and also degrades in culture [36]. Therefore, incorporating collagen into the silk-HRP-crosslinking reactions circumvents collagen contraction issues, while maintaining physiologically relevant cell binding Arg-Gly-Asp (RGD) domains from the collagen [37,38]. Compared to silk-collagen dermal gels, there was a substantially higher amount of contraction within 7 days of culture of the collagen gels (Figure 2A); this difference persisted for the duration of the study (42 days).

The durometer is a tool used to evaluate hardness and viscoelastic properties of materials [28,39]. The mechanical properties of skin are largely influenced by the collagen and elastin fibers located in the dermis, dominated by the important interactions between the fiber network and hydration [40]. However, rarely is the hypodermis directly considered because it is difficult to parse individual layer contributions to skin hardness via the durometer, indentation, nanoindentation, or atomic force microscopy [28,39–43]. It is of interest to develop an in vitro HSE model with mechanical behavior similar to in vivo human skin for improved relevance for certain applications including drug delivery/permeability [16]. While the silk-collagen materials also have significantly higher durometer readings than the explant, the dermal silk-collagen reading is similar to human skin tissue (index pad) found in literature reported as 32 ± 3 (Type O) which would be between 60–70 in Type OO [28] (Figure 2B). This discrepancy could be due to the fact that the explant is obtained from abdominoplasty, and likely has more hypodermis to absorb forces than the index pad [17]. There was not any significant difference between the Dermis (silk-collagen), Dermis (silk-collagen) + Hypodermis combined layers, and the Skin explant, in Stiffness or Young’s modulus (Figure 2C, 2D). However, the Hypodermis by itself is much stiffer than when combined with the dermis, suggesting that the silk-collagen dermis could absorb compressive forces or that energy is dissipated at the interface between the dermis and hypodermis.

With this new biomaterial matrix established, we could address added complexity not normally found in standard HSEs. The most complex clinically and commercially available skin models typically contain at maximum 2 cell types, and are therefore lacking hypodermis, innervation, and immune components. We constructed a multi-layered full thickness in vitro HSE with a nervous system component using only primary human cells. The HSE consists of 3 layers: epidermis, dermis, and hypodermis, including primary human keratinocytes on the epidermis, fibroblasts in the dermis, hiNSCS coating the hypodermis, and numerous cell types within the hypodermis obtained from patient-donated abdominoplasty tissue including adipocytes, pre-adipocytes, smooth muscle pericytes, and endothelial cells [24].

The addition of the hypodermis to HSEs seems to contribute tissue resident immune cells as demonstrated by qRT-PCR and immunohistochemical analyses (Figure S2, Figure 3). The HSE without hypodermis does not have CD68 staining (Figure 3F). We therefore speculate that the addition of the skin to the hypodermis groups could create homeostasis in the tissue (Figure 4) through regulation of glycerol and cytokine secretion sustained with time. In general, there is less overall cytokine secretion from the Skin group—which only contains keratinocytes and fibroblasts—Skin-only groups (keratinocytes + fibroblasts) showed less broad activity overall, with time-depending decreases in MCP-1, IL-6, IL-8, and GRO release. These pro-inflammatory cytokines can be produced by keratinocytes and fibroblasts, and are typically associated with wound healing [44–48]. MCP-1 in human skin is produced by keratinocytes and fibroblasts in response to inflammation or injury to attract monocytes or basophils [47]. It is thought that healthy (fibroblasts and keratinocytes) cells do not produce it unless otherwise stimulated; therefore, it may be possible that the addition of the hypodermis stimulates the HSEs as the signal decreases after D7 (Figure 4A) [49]. MCP-1 is also thought to be related to neural injury response and plays a role in the nervous system as well as the immune system with overexpression implicated in nociceptive pain [50]. In adipose tissue, MCP-1 is produced by normal adipocytes at low levels and overexpression is correlated with obesity [51]. Interestingly, Patient A had a BMI of 74 (morbidly obese) and exhibited MCP-1 expression that was about double (243.5% versus 107.5% of positive control) that of Patient B (BMI 44, obese). IL-6 levels in skin cells, adipose tissue, and in the nervous system have been linked to inflammatory response [44,45,52,53]. Macrophage colony stimulating factor, MCSF-1, is known to be produced by fibroblasts, endothelial cells, smooth muscle cells, and macrophages, among others, at steady state [54]. Recently it has been suggested that lower colony stimulating factor signal could be related to inflammatory conditions, with several clinical trials exploring MCSF-1 (and granulocyte-macrophage colony stimulating factor (GM-CSF)) role in inflammatory arthritis [54,55].

In general, the trends per cytokine secretion were different with respect to each patient (lipoaspirate source). Of note, where the separate components (i.e. Skin or Hypodermis) exhibit certain individual trends, when they were combined (i.e. Skin + Hypodermis, ± Nerve) the effect was not additive, but rather the trends seemed to be controlled by interactions in a system-specific manner. For example, in Patient B (Figure 4A) the Hypodermis exhibited a moderate IL-6 signal at D7 (510% of positive control), the Skin IL-6 level was very low (46.7% of positive control), but the Skin + Hypodermis IL-6 signal was higher than the Hypodermis alone and higher than the individual levels of both Hypodermis and Skin IL-6 combined (685% of positive control).

This large-scale interaction between the keratinocytes, fibroblasts, hiNSCs, and the hypodermal layer resulted in a pattern of initially (D1) high cytokine secretion that diminished by the completion of the study (D42). Neither the Hypodermis nor the Skin control groups had trends consistent between patients, but the Skin + Hypodermis group was consistent (with peaks at D7 for both patient groups). Through the addition of the skin with the hypodermis, with and without nerves, there was an apparent interaction between these components that dynamically affected cytokine secretion. Likewise, via proteomics, similar trends were observed; the addition of the skin, hypodermis, and neuronal components resulted in higher complexity in the proteins identified and quantity of protein detected. The prominence of certain proteins also changed between each group and patient (source of lipoaspirate), but this was consistent with the cytokine analysis, where the HSE with nerves had the highest protein content at D1, decreasing with time. This decreasing trend was not consistently observed with other groups (i.e. Skin, Skin + Hypodermis, Hypodermis controls), which further suggested the importance of including neuronal and hypodermal components in the HSEs.

5. Conclusions

The HSEs described herein, composed of a cross-linked silk-collagen hydrogel as the dermal biomaterial, were more resistant to degradation and contraction over time compared to collagen-only systems. Further, mechanically the silk-collagen HSEs were more similar to human skin explants than collagen alone. The neuronal component of the HSE, as well as the addition of the hypodermis and all the cell types introduced from the lipoaspirate (including adipocytes, pre-adipocytes, endothelial cells, smooth muscle cells, and macrophages) produced an immune-competent tissue capable of tracking inflammatory responses with time. The HSE models that included nerves had more proteins detected compared to the controls. Further, differences observed between patient (lipoaspirate sources) groups, specifically via LC-MS/MS analysis and cytokine secretion, lends the model to potentially be a patient-specific tool for evaluating inflammation.

The innervated, full thickness HSE demonstrated use for long-term culture up to 6 weeks, where the addition of skin with adipose and neuronal layers allows for communication in a time-dependent manner. Pro-inflammatory factors appear to be stable after 7 days. The model is a major improvement upon collagenous HSEs with only skin cells, and can be considered for specific questions related to long-term immune and neuronal activity in the cutaneous system. Ultimately, this system could be utilized for in vitro studies with potential impact on conditions where interactions between the cutaneous, immune, and neuronal system are especially important (e.g. psoriasis).

Supplementary Material

supplement

Figure S1. Mass loss of acellular silk-collagen and collagen-only gels with respect to time.

Collagen gels lost approximately 3.14 more of their initial mass within 72 hours compared to silk-collagen gels (□ silk-collagen, Δ collagen-only). Error bars are ± SEM.

Figure S2. qRT-PCR analysis on hypodermis samples at 1, 3, and 6 weeks culture post-surgery demonstrated elevated markers for macrophages.

Hypodermal-only samples had increasing macrophage markers (CSF1 and CD68) through 6 weeks; pro-inflammatory IL-6 increased with time (in agreement with cytokine secretion observations) as did RANTES, but NOS2 and ACRP30 (adipose marker) decreased with time.

Table S1. Cell culture media conditions.

Table S2. Media conditions for HSEs.

Table S3. Timing of media type with HSEs.

Table S4. Collagen and silk-collagen gels composition.

Table S5. Immunohistochemistry antibody source and concentration.

Table S6. PCR primers.

Table S7. Cytokine nomenclature.