Data on bone marrow stem cells delivery using porous polymer scaffold

Low bioavailability and/or survival at the injury site of transplanted stem cells necessitate its delivery using a biocompatible, biodegradable cell delivery vehicle. In this dataset, we report the application of a porous biocompatible, biodegradable polymer network that successfully delivers bone marrow stem cells (BMSCs) at the wound site of a murine excisional splint wound model. In this data article, we are providing the additional data of the reference article “Porous polymer scaffold for on-site delivery of stem cells – protects from oxidative stress and potentiates wound tissue repair” (Ramasatyaveni et al., 2016) [1]. This data consists of the characterization of bone marrow stem cells (BMSCs) showing the pluripotency and stem cell-specific surface markers. Image analysis of the cellular penetration into PEG–PU polymer network and the mechanism via enzymatic activation of MMP-2 and MMP-13 are reported. In addition, we provide a comparison of various routes of transplantation-mediated BMSCs engraftment in the murine model using bone marrow transplantation chimeras. Furthermore, we included in this dataset the engraftment of BMSCs expressing Sca-1+Lin−CD133+CD90.2+ in post-surgery day 10.


Specifications
This data allows the scientific community to elucidate the penetration mechanism of cells into biomaterials.
This data elucidates the site-specific homing and engraftment of transplanted stem cell chimeras using different routes of administration.

Data
We designed and synthesized a stable, biocompatible, pH-sensitive and enzymatically biodegradable castor oil-based porous polyethyleneglycol-polyurethane (PEG-PU) networks as scaffolds for delivery of BMSCs at an injury site that accelerates the wound tissue repair process ( $ 50% faster) [1]. In this data, BMSCs isolated from murine bone marrow were characterized ( Fig. 1A and B). Cells cultured in presence of polymer network were evaluated microscopically (Fig. 2) and biochemically for MMP-13 and MMP-2 activity (Fig. 3). Next, we utilized an in vivo syngenic murine excisional splinting wound model to evaluate the accelerated wound healing capacity of BMSCs when transplanted using polymer networks as scaffolds (Fig. 4). This dataset also depicts the histological analysis of wound tissue (Fig. 5). This data contains bone marrow transplantation chimeras (male cells in female mouse) for homing at wound site as well as different tissues (Fig. 6). Finally, immunostaining analyses of wound tissue sections to evaluate engraftment of transplanted BMSCs are included (Fig. 7).

Isolation and characterization of mouse BMSC
C57BL/J6 mice were used for the isolation of BMSC. Briefly, bone marrow from tibias and femurs of 8 weeks old C57BL/J6 mice were flushed out using αMEM. Subsequently, cells were plated using the same medium containing 10% FBS and 1% penicillin-streptomycin for 72 h with repeated changes of medium and subsequently passaged to perform experiments [2]. These cells were characterized using RT-PCR for pluripotency and stem cell markers Oct-4, Sox-2, Klf-4, c-Myc along with nestin, CD49f, CD29, CD73, CD44 and Sca-1 gene expressions. 18 S rRNA expressions have been used in the same sample as an internal control [3]. Flow cytometry analyses were also performed for various surface protein markers expression such as CD133, CD44, CD29, CD34, CD106, CD140a, Sca-1, CD11b, Ter119 and Lin. The above mentioned protocol was approved by the Institutional animal ethics committee (approval No. IICT/CB/AD/25/06/2014/13).

Cell penetration of polymer network assay
MDA-MB-231 (breast adenocarcinoma) cells and BMSCs were cultured in 24-well plates with a density of 1 Â 10 5 cells/well. Cells were labeled with deep red tracker dye (Molecular Probes, USA) as described in manufacturer's protocol. The cells were harvested and cultured in presence of PEG-PU for 24 h. The polymer networks were removed, washed and fixed with 4% paraformaldehyde. After embedding in sucrose, 30 mm sections were cut using cryotome [4]. Images were taken under fluorescence microscope (Olympus).

Zymography
MDA-MB-231 (breast adenocarcinoma) cells and BMSCs were cultured in presence and absence of PEG-PU for 24 h in a serum free medium at a cell density of 5 Â 10 5 /well. Conditioned media was extracted and gelatin as well as collagen zymography was performed using the extracts at non reducing conditions as described earlier [5]. Gelatinolytic activity of MMPs was quantified using NIH ImageJ software.  2.4. Excisional wound splinting mouse model 8-10 weeks old C57BL/6J mice were used for generation of excision wound splinting model as described earlier [6]. Mice were anesthetized using an intraperitoneal (ip.) injection of sodium pentobarbital (50 mg/kg). The hair on dorsal side was removed by applying hair removal cream followed by disinfection of skin surface with povidine-iodine solution. Two symmetrical full-thickness excisional wounds were created besides the midline using 5 mm diameter sterile biopsy punch. Transplantation of BMSCs was performed by injecting intradermally (id.; 0.7 Â 10 6 cells) and on the wound surface (0.3 Â 10 6 cells). In a separate group, BMSCs were cultured in presence of 5 mm diameter polymer network and implanted as described above along with placing of the polymer network on the wound surface. A similar 5 mm diameter punched silicon splint ring was adhered around the wound and stitched at the corners to prevent the wound healing due to contraction of the skin and wound was dressed with transparent bandage. The above mentioned protocol was approved by the Institutional animal ethics committee (approval No. IICT/CB/AD/26/08/13/08).

Histopathology studies
Regenerated wound tissue samples from post-surgery day 7 and 10 of control wound, Vehicle control (PEG-PU), transplanted BMSC and BMSC-polymer network mice using 5 mm biopsy punch were fixed in 4% paraformaldehyde. The wounds were mounted on cryo-block using optimal cutting temperature (OCT) compound to make sections of thickness 10 mm using cryotome (Leica, Germany).

Hematoxylin and eosin staining
Cross-sections of skin were mounted on slides and fixed using cold acetone for 10 min. After rehydration for 30 mins, sections were stained with hematoxylin for 5-15 mins and subsequently de- stained with 0.5% glacial acetic acid (if over stained) and washed. Sections were then incubated with eosin for 1-2 min followed by washing with 100% alcohol. The slides were washed with xylene and mounted for imaging under microscope [4].

Sirius red staining
The sections were stained with Sirius red for 30 min to evaluate collagen deposition at the regenerated wound site. The stained sections were washed under running tap water for 2 min. The slides were counter stained with haemotoxylin, washed in xylene and mounted [7].

Immuno-flouroscence analysis
The frozen sections were fixed with cold acetone and dried for 30 min. The slides were washed with PBS and incubated in normal goat serum (1:10 dilution) for 1 h. After blocking, the blocking buffer was drained and incubated with antibodies CD133-PE, CD90.2-APC, Sca-1-PE and Lin-PE for overnight at 4°C. The slides were washed in PBST to remove unbound antibodies. The slides were subsequently washed in xylene and mounted using DPX mounting medium to view under confocal microscope (Olympus FluoView, Japan) [8].

Engraftment of BMSCs
BMSCs were isolated from male C57BL/6J mice and transplanted to female C57BL/6J mice through various routes of administrations such as intradermal, intravenous and/or along with the PEG-PU scaffolds at the wound site (n¼ 5). BMSCs were cultured along with PEG-PU before implantation on wound as described earlier. 7 days post-surgery, wound tissue, along with other tissues/organs such as heart, liver and lungs were harvested from these mice followed by grinding in liquid nitrogen. DNA was isolated from the tissue using NucleoSpin tissue according to manufacturer's manual (Machery-Nagel, Germany). In a separate set of experiments to plot the standard curve, DNA was extracted from isolated BMSCs of male and female C57BL/6J mice, simultaneously. Real time PCR was performed for Y-linked zinc finger protein (Zfy-1) gene. Bcl-2 was used as reference control. Standard curve for the engraftment of male cells was plotted by increasing the % of male BMSC DNA with female BMSCs DNA as described earlier [9]. Zfy-1 gene expression level was calculated as the value of 2 À δ Ct. Percent of male cells in various tissues of female mice was calculated using the standard plot [9].

Image analysis
The image files were opened using ImageJ software followed by conversion in 16-bit by various sequential steps provided in the software: Edit -Options -Scale. The staining was quantified by adjusting the threshold in the following steps: (I) "Image -Adjust -Threshold" -The auto/manual setting was used to select all the stained portions, (II) Processto subtract background with rolling ball thenapply, (III) Processbinarywatershed, (IV) using "Analyze -Set Measurements" options finally selected the parameters to be measured. To make sure that only the selected gray level measurements are quantified, "Limit to Threshold" option was used, (V) "Analyze -Measure," results appeared in a table form was saved and graphs were made by transferring this data to excel file, (VI) "Analyze -Analyze Particles" hava been used to measure individual feature profiles. Intensity measurements are performed within regions of interest by choosing the parameters at step V [10].