Experimental datasets on processed eggshell membrane powder for wound healing

Eggshell (ES) and eggshell membrane (ESM) is a significant byproduct of the egg producing industry (Ahmed et al., 2019). Many studies have been undertaken to utilize ES waste for potential value added applications (Cordeiro and Hincke, 2011). Described here are the datasets from our evaluation of processed eggshell membrane powder (PEP) as a wound healing product using the mouse excisional wound splinting model (Ahmed et al., 2019). PEP biomaterial was characterized by proteomics using various extraction and solubilization strategies including moderate (lithium dodecyl sulphate (LDS) and urea/ammonium bicarbonate) and harsh conditions (3-mercaptopropionic acid (3-MPA) and NaOH/dimethylsulfoxide) in order to progressively overcome its stable, insoluble nature (Ahmed et al., 2019, Ahmed et al., 2017). Analysis of proteomic data allowed the relative abundance of the main PEP protein constituents to be determined. The efficacy of PEP for promotion of wound healing was assessed using the mouse excisional wound splinting model, and well-established semi-quantitative histological scoring. (More details about the PEP biomaterial characterization and its in vivo evaluation can be found in the related research article (Ahmed et al., 2019)).

main PEP protein constituents to be determined. The efficacy of PEP for promotion of wound healing was assessed using the mouse excisional wound splinting model, and well-established semiquantitative histological scoring. (More details about the PEP biomaterial characterization and its in vivo evaluation can be found in the related research article

Data format
Raw and analyzed Experimental factors Mass spectrometry: Eggshell membrane collected at the egg breaking unit was processed (washed, milled, sieved, and g sterilized) into a micronized powder (<100 mm), which was designated "Processed Eggshell Membrane Powder" (PEP). For proteomics, PEP samples were subjected to various extraction and solubilization strategies including moderate (via lithium dodecyl sulphate (LDS) and urea/ammonium bicarbonate (NH 4 HCO 3 )) and harsh conditions (via 3-mercaptopropionic acid (3-MPA) and NaOH/ dimethylsulfoxide) conditions. Samples prepared by 3-MPA, NaOH/DMSO, and LDS/DTT treatment were subjected to in-gel digestion, while in the case of urea/NH 4 HCO 3 extraction, in-solution digestion was performed. The protein constituents of PEP were identified using LC/MS/MS analysis, with a false discovery rate (FDR) of 1% and at least two unique peptides. Keratins were discarded from the identified protein inventory. In addition, any protein identified with only one unique peptide (according to the Scaffold software interface) was discarded from the final protein inventory. Tissue processing: The processed wound samples were cut into two halves (Upper and lower halves) and then embedded in paraffin (Leica Biosystems Inc., ON, Canada). PEP (50 mg) was suspended in PBS and centrifuged. The resultant pellet was centered in pre-embedding media and processed with the LOGOS tissue processor. Digital scanning: Stained tissue and PEP sections were scanned with Zeiss Mirax Midi whole slide digital scanner (12 slides/scan and 40X objective lens). Exposure time was 10-100 ms (bright field) and the specimen threshold level of 40-45.

Experimental features
A complete protein inventory for PEP was created by merging the proteins identified by LC/MS/MS analysis after various extraction and solubilization strategies. Relative abundances of proteins identified in the PEP biomaterial were determined using Scaffold proteome software. The effect of PEP on wound healing was evaluated in the mouse excisional wound splinting model using the macroscopic planimetric timecourse (30e38 mice) and a histological scoring system (4 mice each at time points 3, 10, and 17

Data
The presented data demonstrates the utilization of various extraction strategies (moderate to harsh conditions) [1e4] to identify the protein constituents of PEP using the proteomic approach (Table 1). A comprehensive PEP proteome was established and compared to the general ESM proteome ( Fig. 1 and Table 2). LC/MS/MS spectrometry data was interpreted in order to determine the relative abundance of

Value of the data
The presented data describes the utilization of various extraction and solubilization strategies [1e4] to identify the protein constituents of PEP by proteomics. The proteomic approach allows the estimation of relative abundances of the main protein constituents of PEP biomaterial. The data demonstrates the use of animals (C57BL/6J mice) for the planimetric timecourse and histological assessment of healing of the splinted excisional wound after application of a biomaterial (PEP). The data describes an established histological scoring system used to assess the effect of PEP on various histological parameters critical to assess wound healing promotion. The histological processing of PEP biomaterial via pelleting and pre-embedding in agar-formalin media provides researchers with a strategy to process powdered biomaterials and even cells.

Table 1
Various extraction conditions used for the in-solution and in-gel digestion-based proteomic analysis of PEP.

Extraction strategy
In-solution digestion a In-gel digestion      the main protein constituents of PEP biomaterial ( Table 3). The kinetics of wound healing (with and without PEP) in the mouse splinting excisional wound model was determined using a macroscopic planimetric strategy with histological scoring ( Table 4). The histological scoring system was established to assess various histological parameters including degree of angiogenesis, collagen deposition, fibroblast infiltration, macrophage infiltration, polymorphonuclear cells (PMN) infiltration, fibrin clot formation, epidermal differentiation and indentation along with the presence of multinucleated giant cells (Table 5). Finally, PEP was stained with Masson's trichrome to confirm the absence of stainable collagen using an innovative pre-embedding histological approach (Fig. 2).

Relative abundance of PEP protein constituents
MS/MS peak lists were generated using ProteinPilot (Version 4.5) and analyzed using Mascot (Version 2.4.0) and X!Tandem (CYCLONE version), both programmed to search the TAX_GallusGal-lus_9031_20141114 database (unknown version, 222,250 entries). Validation of MS/MS based peptide and protein identification was performed using Scaffold Proteome software (version 4.  spectra were searched against the Uniprot and NCBI chicken databases. The relative abundance of the PEP protein constituents was estimated by averaging the total spectral count of each identified protein using the aforementioned Scaffold Proteome software (Table 3).

In vivo study
All in vivo experiments were conducted following the approved animal protocol (CMM 2108) by the University of Ottawa Animal Care committee and according to the guidelines of the Canadian Council on Animal Care (CCAC). All animal protocols are in compliance with the NIH Guide for Care and Use of Laboratory Animals (Animal Welfare Assurance # A5043-01). Capacity of PEP for promotion of wound healing was assessed using the well-established mouse excisional wound splinting model [5]and the subsequent macroscopic planimetric timecourse [6] and histological scoring. A total of 38C57BL/6J male mice (10e12 weeks old, Jackson Laboratories, USA) were used for the entire study (Table 4).

Histological assessments
PEP (50 mg) was suspended in 1 mL PBS and centrifuged for 5 minutes at 13,000 rpm. The resultant pellet was centered in a base mould; pre-mounting media composed of 2% agar and 10% formalin was poured gently over the pellet and left for few minutes to solidify. The resulted PEP block was processed using the LOGOS tissue processer, embedded in paraffin and then sectioned using a Leica microtome. PEP sections were stained using Masson trichrome to confirm the absence of stainable collagen in the PEP biomaterial (Fig. 2). For evaluation of wound healing, histological scoring system was established to assess parameters that represent wound healing [7e9], including degree of angiogenesis, collagen deposition, fibroblast infiltration, macrophage infiltration, polymorphonuclear cells (PMN) infiltration, fibrin clot formation, epidermal differentiation and indentation along with presence of multinucleated giant cells. Every parameter was given a score of 0e5 based on its graded level of abundance. Score 0 indicates complete absence, while score 5 indicates profound manifestation of the assessed parameter. Scoring of collagen deposition was based, not only on the degree of abundance (i.e. absent, scanty, low, moderate, profound, restored), but also on the degree of organization (disorganized, fragmented, separated, organized) ( Table 5).