Decellularized Wharton's Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications.

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 ± 0.12 μg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

1. Introduction

Disease or trauma to the human body leads to damage and degeneration of tissues, thereby requiring their repair, replacement or regeneration. Tissue regeneration requires an optimal combination of cells, scaffolds, appropriate media and growth factors. The properties of an ideal scaffold for tissue regeneration are high porosity, biocompatibility, biodegradability and mechanical properties consistent with and suitable to the location of implant [1]. Current treatment options for tissue regeneration involve the use of autografts or allografts. Nevertheless, autografts can be difficult to obtain due to expensive and painful procedures, while allografts pose the risk of infection and immune rejection. Several types of scaffolds from natural or synthetic sources (polymers, ceramics, and composites) have been developed for tissue regeneration over the years [1]. Since, such scaffolding is associated with material-specific limitations, there is growing interest in the use of biocompatible, natural bioactives, or synthetic materials as alternatives. [2-8].

Wharton’s jelly (WJ), is a firm mucoid connective tissue surrounding umbilical cord vessels [9], possessing many unique biochemical characteristics required for a scaffold. Mesenchymal stem cells (MSCs) are derived from WJ and immersed in ground substance that is rich in collagen, hyaluronan, and also containing numerous sulfated glycosaminoglycans (GAGs) [9]. MSCs widely express the archetypal hyaluronan receptor, CD44, also expressed on osteocytes, chondrocytes, and hematopoietic marrow cells [10]. WJ is a rich source of peptide growth factors, notably insulin-like growth factor-1 (IGF-1) and to a lesser extent platelet-derived growth factor (PDGF), [11] both of which are linked to controlling cell proliferation, differentiation, synthesis and remodeling of the extracellular matrix [12].

This work is based on a hypothesis that customized cell removal procedures can effectively process WJ to produce a decellularized, bioactive Wharton’s jelly matrix (DWJM). We also postulated that DWJM would provide a 3D environment specifically well suited to support undifferentiated mesenchymal cell culture. This paper details the decellularization processes used to obtain this matrix, in addition to its characterization and analysis of its structural contents. Here, we also demonstrate that WJ and bone marrow MSCs (BMMSCs) can be seeded and cultured in vitro upon this matrix. We further studied the gene expression profiles of these MSCs when seeded on our 3D scaffold, and also assessed the biocompatibility of our matrix in vivo using a murine bone defect model.

2. Materials and methods

Human umbilical cord collection, WJMSCs and WJ tissue harvest followed by decellularization were performed in accordance with the approved University of Kansas Medical Center’s Institutional Review Board protocol # HSC 12129 (title—Decellularization of umbilical cord Wharton’s jelly for tissue regenerative applications including avascular necrosis) at the University of Kansas Medical Center. Consents were collected from donors by obtaining their written signatures on the approved informed consent form. Umbilical cords were immediately collected from consented mothers with full-term pregnancy after normal vaginal delivery. The umbilical cord was placed in a transport solution made of Lactated Ringer’s solution supplemented with penicillin 800 U/mL (Sigma-Aldrich, St. Louis, MO), streptomycin 9.1 mg/mL (Sigma-Aldrich), and amphotericin 0.25 mg/mL (Sigma-Aldrich) and immediately refrigerated at 4°C. The decellularization process was initiated within 72 hours of umbilical cord collection.

2.1 Decellularization process

The decellularization procedure has recently been described in our earlier publication [13]. Briefly, fresh human umbilical cords were transported from the delivery room in a transport solution at 4°C. Umbilical cords were dissected in a laminar flow safety cabinet, by separating the matrix into large oval pieces away from the surrounding membranes and vascular structures. They were then subjected to two cycles of osmotic shock, alternating with a hypertonic salt solution containing sodium chloride, mannitol, magnesium chloride, and potassium chloride with an osmolarity of approximately 1,275 mOsm/L, and against a hypotonic solution of 0.005% Triton X-100 in ddH2O centrifuged at 5,000 rpm at 4°C.

After two cycles of osmotic shock, the tissues were subjected to an anionic detergent (sodium lauryl) and, sodium succinate (Sigma L5777), then switching to a recombinant nucleic acid enzyme, (Benzonase™) in buffered (Tris HCl) water for 16 hours. Following this, an organic solvent extraction with 40% ethyl alcohol was performed for 10 minutes at 5,000 rpm in the centrifuge at 4°C. All of the detergent and other processing residuals were then bound and removed utilizing ion exchange beads (iwt-tmd (Sigma), XAD-16 Amberlite beads (Sigma), and Dowel Monosphere 550A UPW beads (Supelco)) in a reciprocating flow-through glass system at room temperature in ddH2O for 30 hours. The decellularized matrix was cryopreserved using 10% human recombinant albumin (Novozymes) and 10% DMSO (Sigma) solution in standard RPMI media, employing a material-specific computer controlled freezing profile developed to freeze at -1°C/minute to -180°C [14].

2.2 Isolation, expansion, and WJMSCs seeding onto DWJM

a. Preparation of DWJM for seeding with WJMSCs

Freshly obtained fragments of DWJM were transferred to a large petri dish and covered with phosphate buffered saline (PBS). DWJM pieces (5–7 mm in diameter) were obtained using a sterile 5–7 mm skin punch biopsy kit. The resulting DWJM pieces were cylindrical in shape and with non-uniform heights varying between 2–3 mm. DWJM scaffold volume obtained was approximately 72 mm3. From this point on, these pieces of DWJM will be referred to as “DWJM scaffolds”. DWJM scaffolds were transferred using sterile forceps to a large petri dish and washed twice with PBS then transferred to non-tissue culture treated plates at the time of seeding.

b. MSC isolation and expansion

i. WJMSCs—WJMSCs were isolated and expanded according to the procedures described by Wang et al [15]. Briefly, the outer layer of the cord was carefully removed and the cord was cut into smaller segments. The blood vessels were dissected from these cord segments and then cut into smaller pieces and digested with Collagenases (Worthington Biochemical Corporation, Lakewood, NJ) in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) with 10% Fetal Bovine Serum (FBS) (Atlanta Biologics, Atlanta, GA) and 1% penicillin/streptomycin (Sigma-Aldrich) overnight at 37°C to obtain WJMSCs. The WJMSCs were passaged and maintained in this low glucose DMEM-10% FBS-1% penicillin/streptomycin medium with passages 4–9 being used for the following experiments.

ii. BMMSCs—BMMSCs were isolated from bone marrow aspirates of healthy consented donors at University of Kansas Medical Center (HSC # 5929). The cells were isolated following standard ficoll density gradient separation method (Lymphoprep, Stem Cell Technologies, Vancouver, BC). The isolated cells were maintained in high glucose DMEM (Sigma-Aldrich), 20% FBS (Atlanta Biologics) and 1% penicillin/streptomycin (Sigma-Aldrich) at 37°C, under 5% CO2 and 90% humidity.

c. MSC characterization and phenotyping

The MACS Miltenyi Biotec MSC human phenotyping kit was used to characterize the expanded WJMSCs and BMMSCs, and analyzed by BD Flow Cytometer LSR2 (Beckton Dickinson). MSCs isolated from human umbilical cord and bone marrows were stained for CD14, CD20, CD34, CD45, CD73, CD90 and CD105.

d. MSC seeding onto DWJM

For each set of seeding experiments, Single-donor (n = 1) WJMSCs or BMMSCs were used. 1 x 106 MSCs suspended in 50-μL culture medium were seeded on each DWJM scaffold (average seeding density 1.4 x 104–4 x 104/mm3 per DWJM scaffold) in a 48-well plate, followed by addition 1 mL of culture medium per well.

For the gene expression studies, 0.25 x 106–1 x 106 WJMSCs or BMMSCs from passages 4–9 were seeded on DWJM in a 24-well non tissue culture treated plate (Corning Inc., Corning, NY) for 4 to 7 days and cultured in their respective media.

2.3 Characterization of DWJM

a. DNA quantification

DNA in the samples was isolated using the Qiagen DNeasy Blood and Tissue Kit (Dusseldorf, Germany) according to the manufacturer’s instructions. Pico Green dye (Molecular Probes, Eugene, OR) was used as a label and the extracted DNA quantified fluorometrically using Quant-iT dsDNA HS (high sensitivity) Kit (Invitrogen, Carlsbad, California). The amount of extractable DNA was calculated as wet weight tissue and expressed as a percent reduction in extractable DNA relative to that for non- decellularized tissue. All analyses were run in triplicate.

b. Glycosaminoglycan’s (GAGs) content analysis

The Blyscan assay (Biocolor Life Sciences, UK) was used according to the manufacturer’s instructions for analysis of sulfated glycosaminoglycan content. Tissue samples from native umbilical cord WJ and DWJM were analyzed and the results were reported as μg/mg of glycosaminoglycan per wet tissue weight.

c. Protein identification by mass spectrometry

DWJM samples from two different umbilical cords samples were analyzed following two methods of protein extraction. For the first method, DWJM was snap-frozen using liquid nitrogen, tissue homogenized, and suspended in WJMSC culture medium as described above.

The second method used the Ready Prep Protein Extraction Kit with zwitterion detergent ASB-14 as a solubilizing agent (Bio-Rad Laboratories, Inc., Hercules, CA). This step was followed by a cleanup step to free the proteins of ionic contaminants by selective precipitation of detergents, lipids using Ready Prep 2-D Cleanup Kit (Bio-Rad Laboratories, Inc., Hercules, CA). The protein pool present in the extracts was denatured in 6M guanidine hydrochloride, reduced, alkylated, and subsequently digested for 18 h with sequencing grade trypsin (12 ng/L, Promega, Madison, WI) at 37°C. Following enzymatic digestion, the extracted peptides were concentrated to a final volume of 50 μL on a Centrivac Concentrator (Labconco, Kansas City, MO). The peptide extracts were analyzed by reversed phase chromatography using a 2D NanoLC HPLC (Eksigent Technologies, Dublin, CA) coupled to a Linear Thermo Electron Quadripole Electron Trap—Fourier Transformation (LTQ FT) mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The mass spectrometer was controlled by the Xcalibur software to perform continuous mass scan on the FT in the range of 400–1900 m/z at 50,000 resolutions, followed by MS/MS scans on the ion trap of the six most intense ions. All tandem mass scans were searched using the Proteome Discoverer (version 1.3, Thermo Fisher) against a human protein database using trypsin cleavage specificity, with a maximum of 2 missed cleavages. The following variable modifications were selected: oxidation of M (methionine), deamidation of N (asparagine), and Q (glutamine), and carboxymethylation of C (cysteine) selected as fixed modifications, with a maximum of 4 modifications/peptides allowed. Estimation of false discovery rate (FDR) was conducted by searching all spectra against a decoy database. For protein identification an FDR >1% (high confidence) was defined for all peptides of interest, which were subsequently reviewed manually.

2.4 Evaluating seeded WJMSC adherence to and penetration of DWJM scaffolds

a. Confocal microscopy

To assess WJMSCs attachment to DWJM scaffolds after cell seeding and culture, scaffolds were transferred to a viewing chamber for confocal microscopy examination using a Fluoview scanning laser confocal microscope (Olympus, Center Valley, PA). Prior to viewing, the seeded DWJM scaffolds were rinsed twice with PBS and incubated with 1 mL culture medium containing 2 μg Calcein stain (Molecular Probes, Eugene, OR). Calcein is a cell-permeant dye that is converted to green-fluorescent Calcein when in live cells. Using this stain, we tracked live WJMSCs after their being seeded onto DWJM scaffolds at 2, 24 and 48-hour intervals.

b. Dual beam electron microscopy

MSCs were seeded on DWJM as described above and cultured in their appropriate media for 7 days. The matrix with cells was collected after 24 hours and at day 7 and was fixed overnight in 4% paraformaldehyde (VWR, Randor, PA) in PBS at 4°C. Tissue specimens were washed three times in PBS then stained with 2% osmium tetroxide (OT) for 24 hours to label lipids. Since OT gives off a strong electron backscatter signal, OT- stained samples were again washed three times for 10 minutes in PBS. All samples were gradually dehydrated with ethanol and cleared in xylene, before being embedded in paraffin. Samples were sectioned to a thickness of 10 μm or 20 μm using a microtome (Leica, Buffalo Grove, IL) and mounted on Super Frost glass slides (Thermo Fisher, Waltham, MA). OT-stained samples were deparaffinized using two 3 minute washes of xylene and critical-point dried in 100% ethanol using an Autosamdri 815B super-critical dryer (Tousimis, Rockville, MD) Samples were sputter-coated with 5 nm of copper using a Q150T Turbo-Pumped Sputter Coater (Quorum Technologies, West Sussex, and United Kingdom) and then imaged on a Versa 3D Dual Beam electron microscope (FEI, Hillsboro, OR) at a voltage of 30 kV. An Everhart-Thornley detector (ETD) and circular backscatter (CBS) detectors were used to detect secondary electrons and backscatter electrons, respectively.

c. Live cell imaging

DWJM scaffolds of 30μ thickness were placed in a 12 well plate and the matrix blocked with 3% BSA for 2 hours and subsequently incubated with 3 μL anti-fibronectin antibody [F1] (Alexa Fluor® 488) (Abcam, Cambridge, MA) in the dark for 12–15 hours at 4°C. Immediately prior to being seeded onto DWJM scaffolds, the WJMSCs were cultured in a 12 well plate and labeled using the CellVue® Burgundy Labeling Kit (Affymetrix eBioscience, Santa Clara, CA) according to the manufacturer’s instructions. Briefly, 2.5–5 x 105 WJMSCs were seeded on the labeled matrix, and cultured for 24 hours at 37°C. Imaging was made on a Leica 10X HC PL Fluotar 506505 objective of a semi-automated Leica DMIRE2 inverted epifluorescent microscope outfitted with a Ludl Bioprecision motorized stage, Sutter Instruments Xenon Lamphouse with shutters and a Retiga SRV CCD camera controlled by customized TiLa KU (KU Time Lapse) acquisition and image processing software. Regions under study/interest were imaged in Bright field, GFP (Chroma 41001 HQ480/40 excitation HQ535/50 Emission) and CY5 (Chroma 49006 ET620/60 excitation, ET700/75 emission). DWJM interaction with WJMSCs was imaged at 15-minute intervals over an 18-hour period.

d. Scanning electron microscopy (SEM)

The DWJM scaffolds were fixed in 2% glutaraldehyde for SEM processing. The fixed samples were washed with PBS for 10 minutes, placed into buffered 1% osmium tetroxide for 1 hour, and then washed 3 times each for 10 minutes in distilled water. The samples were then dehydrated through a graded series of ethanol at concentrations 30%, 70%, 80%, 95%, and 100% for 15 minutes each. Following this, the samples were critical point dried in CO2 in a model EMS 850 dryer (Electron Microscopy Sciences, Hatfield, PA), then mounted onto aluminum and coated with gold in a Pelco SC-6 sputter coater. The samples were finally viewed using a Hitachi S-2700 scanning electron microscope.

e. Transmission electron microscopy (TEM)

Scaffolds for TEM were rinsed in a buffer prior to fixing in 1% to 2% osmium tetroxide for 1 hour. After osmication, the scaffolds were dehydrated in an ethanol series of 30%, 70%, 80%, 95%, and 100%, 10–15 minutes for each concentration, then placing them in propylene oxide (PO) twice for 10 minutes. To enhance tissue infiltration, the scaffolds were then placed in an equal mixture of resin and PO overnight. At this point, the 1:1 mixture mix was removed from the tissue and fresh 100% resin mixture added to the sample, and allowed to sit on a platform rocker for at least 30 min. The samples were subsequently covered in resin and finally were placed in 60°C oven overnight to cure the resin. The samples were sectioned afterwards using a Leica UCT ultra microtome slicing at 80 nm in thickness, contrasted with 4% uranyl acetate and Sato's Lead Citrate and viewed at 80 KV with a JOEL JEM-1400 TEM.

2.5 Histology and immunohistochemistry

DWJM scaffolds were fixed in either 10% formalin or 4% paraformaldehyde, embedded in paraffin, sectioned, and stained. Slides were reviewed using an Olympus BX40 microscope and pictures were acquired using a DP72 digital camera (Center Valley, PA).

a. Gomori’s trichrome staining

Tissue sections were stained with trichrome stain kit—Richard Allan 87020 (Thermo Fisher, Waltham, MA) according to the manufacturer’s instructions. Using this stain, the nuclei were stained bluish-black to black, cytoplasm, muscle fibers and keratin stained red, while collagen and mucus stained blue.

b. Immunohistochemistry

Immunohistochemistry staining was performed at room temperature using an IntelliPATH FLX™ automated stainer (Biocare Medical, Concord, CA) at room temperatures. Briefly, after deparaffinization, sampleswere blocked in 3% hydrogen peroxide for 10 minutes, rinsed and blocked in strepatividin/biotin (Vector Laboratories, Burlingame, CA.). The samples were again rinsed and stained for 60 minutes at room temperature with 1:200 dilution hyaluronic acid binding protein (RMD Millipore, Danvers, MA) followed by a 15 minute labeling with horse radish peroxidase (HRP) (Dako, Carpinteria, CA). Chromogenic detection of the enzyme conjugate was made using 3,3’ diaminobenzidine (DAB) (DAkp, Carpinteria, CA) when applied for 5 minutes with slides counterstained with hematoxylin.

For collagen immunohistochemistry, after deparaffinization and rehydration, the tissue sections were incubated with a primary antibody against Collagen I (Abcam, Cambridge, MA). Following a 30-minute incubation at room temperature and rinsing, the sections were incubated with secondary antibody, goat anti-rabbit HRP-polymer MACH 2 rabbit HRP-polymer (Biocare Medical, Concord, CA). Finally, collagen staining was visualized by DAB (Dako, Carpinteria, CA) and the nuclei as counterstained by hematoxylin.

Green fluorescent protein (GFP) immunohistochemistry staining was performed by incubation with primary monoclonal goat-anti GFP antibody (1:100 dilution for 45 minutes), followed by secondary MACH2 rabbit antibody (CST, Cell Signaling Technology, Danvers, MA) MACH 2 for 45 minutes on a Clinical intelliPATH FLX automated slide stainer.

2.6 Evaluating seeded WJMSC proliferation

The alamarBlue® (AB) cell viability assay (ThermoFisher Sci, Waltham, USA) was used to assess WJMSC viability and proliferative response following seeding onto DWJM by correlating fluorescent or absorbance signal related to metabolic activity. DWJM scaffold pieces (7 mm in diameter and 2–3 mm in height) were seeded with the expanded human WJMSCs at 1 x 106 cells onto each DWJM scaffold. For controls, 1 x 106 cells per well were cultured as a monolayer in each well of a 24-well plate. AB was assessed at 24 and 48 hours as well as 1 week following WJMSC seeding. These experiments were performed in triplicate.

2.7 Evaluating WJMSC migration toward DWJM by the trans-well migration assay

WJMSC suspension at 3–7 x 105 was loaded to the upper chamber of Transwell set (Costar, Corning Inc.) and the minced DWJM tissue in low glucose DMEM with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin added to the lower chamber. After 4 hours, the trans-wells were removed and the migrated cells counted for viability using a Vi-cell (Beckman-Coulter). All the experiments were conducted in triplicate.

2.8 Molecular studies

a. RNA extraction from cells

WJMSCs and BMMSCs were cultured as a monolayer (2D) or on DWJM (3D) as described in section 2.2d for 7 days. The cells were collected at day 0 (monolayer prior seeding DWJM), day 4 and day 7 after seeding onto the matrix. WJMSCs were harvested from the scaffolds following overnight digestion with Collagenase II. The MSCs were washed twice with PBS and centrifuged at 13000 rpm, for 20 minutes at 4°C to obtain a cell pellet which was suspended in 1 mL monophasic phenol guanidine isothiocyanate (Trizol) (Life technologies) and stored at -80°C until further processing. Once all the samples were collected, RNA was extracted using the standard procedure according to the manufacturer (Life Technologies, Carlsbad, CA).

Briefly, the RNA was separated from the aqueous phase using 0.2 mL chloroform, then 0.7 volumes of isopropanol added with centrifugation to precipitate RNA. The RNA pellet was washed twice with 75% ethanol and dissolved in 30–50 μL nuclease-free water. RNA was quantified by Nano drop spectrophotometer 8000 (Thermo Fisher Scientific). First, 1.5μg of RNA was treated and isolated using the DNA-free™ DNase l kit (Life Technologies). Then a high capacity cDNA reverse transcription kit (Applied Biosystems) was used to generate cDNA from the extracted total RNA samples by a Bio-Rad T100 thermal cycler.

b. Quantitative real-time PCR analysis

The quantitative real-time PCR (qPCR) reactions (20 μL) were performed with the TaqMan gene expression master mix (Life Technologies), and TaqMan array 96 well plates (Applied Biosystems, Foster City, CA) using the StepOnePlus™ real-time PCR system (Applied Biosystems). The primers used are described in Table 1. The qPCR reactions were performed in triplicate. StepOnePlus™ real-time PCR system (Applied Biosystems) was used for qPCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize the samples to obtain cycle delta threshold over background (ΔCT). The delta delta CT method (2-ΔΔCT) was used to analyze the relative gene expression levels.

Table 1

Real time RT-PCR TaqMan primers and their description.

Gene symbol	Detector	Gene name
GAPDH	GAPDH-Hs99999905_m1	Glyceraldehyde-3-phosphate dehydrogenase
ACAN	ACAN-Hs00153936_m1	Aggrecan
SOX9	SOX9-Hs00165814_m1	SRY (sex determining region Y)-box 9
COL2A1	COL2A1-Hs00264051_m1	Collagen, type II, alpha 1
ALPL	ALPL-Hs01029144_m1	Alkaline phosphatase, liver/bone/kidney
RUNX2	RUNX2-Hs00231692_m1	Runt-related transcription factor 2
ITGB1	ITGB1-Hs00559595_m1	Integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)
CD44	CD44-Hs01075861_m1	CD44 molecule (Indian blood group)
THY1	THY1-Hs00174816_m1	Thy-1 cell surface antigen
ENG	ENG-Hs00923996_m1	Endoglin
ALCAM	ALCAM-Hs00977641_m1	Activated leukocyte cell adhesion molecule
CD14	CD14-Hs00169122_g1	CD14 molecule
MKI67	MKI67-Hs01032443_m1	Antigen identified by monoclonal antibody Ki-67
BAX	BAX-Hs00180269_m1	BCL2-associated X protein
VIM	VIM-Hs00185584_m1	Vimentin
ACTA2	ACTA2-Hs00426835_g1	Actin, alpha 2, smooth muscle, aorta
SPP1	SPP1-Hs00959010_m1	Secreted phosphoprotein 1
COL1A	COL1A1-Hs00164004_m1	Collagen, type I, alpha 1
COL4A1	COL4A1-Hs00266237_m1	Collagen, type IV, alpha 1
COL6A1	COL6A1-Hs01095585_m1	Collagen, type VI, alpha 1
DES	DES-Hs00157258_m1	Desmin
HAS2	HAS2-Hs00193435_m1	Hyaluronan synthase 2
BGN	BGN-Hs00156076_m1	Biglycan
VCAM1	VCAM1-Hs01003372_m1	Vascular cell adhesion molecule 1
NOS3	NOS3-Hs01574659_m1	Nitric oxide synthase 3 (endothelial cell)
PCNA	PCNA-Hs00427214_g1	Proliferating cell nuclear antigen

2.9 Animal studies

2.9.1 Animal surgeries

Prior to the animal studies, DWJM scaffolds were washed twice in PBS and pre-incubated in low glucose DMEM (Sigma-Aldrich) with 10% FBS (Atlanta Biologics) and 1% penicillin/streptomycin (Sigma-Aldrich) for 24 hours at 37°C, 5% CO2 and 90% relative humidity. After incubation, DWJM scaffolds were washed multiple times in PBS to remove excess media before transplantation. All the animal experiments were performed in strict accordance with the approved University of Kansas Medical Center Institutional Animal Care and Use Committee (IACUC) protocol # 2013.2158. All the animal studies were performed on transgenic 10kB DMP1—Cre floxed mice (6–8 week old) expressing green fluorescent protein (GFP)[16]. Mice were anesthetized with intraperitoneal injection (IP) of ketamine (90–150 mg/kg) (Vedco) and xylazine (7.5–16 mg/kg). Buprenorphine SR (0.15–0.5 mg/kg) (Zoopharm pharmacy) was given subcutaneously immediately before the surgical procedure for analgesia. One midline skin incision of approximately 1cm in length was made on the dorsal surface of the cranium, followed by the separation of skin and periosteum. A full-thickness parietal bone defect (5.0-mm in diameter) was implemented with a trephine bur (Fine Science Tools, Foster City, CA) attached to an electric Dremell hand piece (Ideal micro drill, Harvard apparatus, Holliston, MA). The defect was left empty (n = 4) or filled with the decellularized matrix (n = 4) and the skin incision was sutured with 5–0 coated Vicryl (polygalactin 910) (Ethicon™, Johnson and Johnson Co., New Brunswick, NJ). Heat was provided during the entire procedure and recovery by circulating warm water blankets to protect the animals from hypothermia. Post—surgery, the animals were monitored for signs of abnormal behavior, paralysis, or infection at the surgical site such as swelling, redness and discharge, and checked daily for 3 days, followed by once every other day for the duration of the study. Animals with cranial defects, and not receiving an implant served as controls. Craniotomy defects in mice were either left un-implanted (control) or implanted with DWJM to study the cellular migration and localization by observing GFP expression. The mice were humanely sacrificed after 14 days by carbon dioxide euthanasia as the primary method of euthanasia, followed by decapitation as the secondary method in accordance with the above-mentioned institutional IACUC protocol.

2.9.2 In vivo IVIS imaging

Mice were anaesthetized with isofluorane gas prior to imaging at 24 and 48 hours by an IVIS station (Perkin Elmer), and then euthanized according to protocol.

2.9.3 Tissue samples

Cranial samples with the matrix were collected in 10% phosphate buffered formalin (Newcomen Supply) within 24 hours. Tissue specimens for the animal study were decalcified using the Rapid Bone Decalcifier solution (American MasterTech) for 5–10 minutes, paraffin embedded, sectioned vertically, and stained as described above.

2.10 Statistical analysis

All data were expressed as means ± standard error of mean (SEM) using a threshold of p ≤ 0.05 determined statistically significant, and analyzed by the Student’s t-test, two-way analysis of variance (ANOVA), with post-hoc Bonferroni, or non-parametric Man-Whitney U testing. A threshold of p ≤ 0.05 determined statistical significance. The statistical analyses were performed utilizing Graph Pad Prism software version 6 (Graph Pad Software, Inc.).

3. Results

3.1. Characterization of DWJM scaffold structure, biochemical components, and biomechanics

DWJM scaffolds were prepared from the isolated and decellularized matrix as shown in Fig 1A. Various methods were used to test the effectiveness of the decellularization process for these scaffolds. Histologically: DWJM was porous and devoid of intact cells, nuclei or other cellular components (Fig 1B). Trichrome staining of the human umbilical cord (Fig 1D) shows collagen-rich extracellular matrix in blue, nuclei/cells in dark blue/black distributed in the matrix and blood vessel wall, and blood in red. Since blood vessels are removed and the cells are lost during the decellularization process, the matrix obtained is rich in blue stain representing collagen (Fig 1E). Immunohistologically: staining for collagen (Fig 1C) and hyaluronic acid (Fig 1F) demonstrate that DWJM is rich in collagen and hyaluronic acid (Fig 1C and 1F). Scanning electron microscopy imaging: indicates that DWJM has interconnected open spaces, with sizes ranging from 20 to 100 μm (Fig 1G). Transmission electron microscopy: demonstrates an absence of intact cells the DWJM scaffold under examination (Fig 1H). Thus, the decellularization process resulted in a porous matrix, rich in collagen and hyaluronic acid and devoid of cells.

Fig 1

Characterization of Decellularized Wharton’s Jelly Matrix (DWJM).

A) A fragment of the isolated DWJM. A skin punch biopsy kit, (right lower corner image) was used to obtain 5–7 mm DWJM scaffolds. B) hematoxylin and eosin (H&E) stained sections of the DWJM showing empty spaces. (Scale bar represents 0.1 mm.) C) Collagen I immunohistochemistry of the DWJM (scale bar is 50 μm), D) Trichrome staining images of human umbilical cord, and E) Decellularized Wharton’s jelly matrix. (Scale bar represents 50 μm.) Red color represents blood, light blue collagen, and cells/nuclei are in black/dark blue. F) Immunohistochemical staining of DWJM by anti- hyaluronic acid antibody. The matrix is rich in collagen and there is abundant hyaluronic acid expression at some parts compared to the others. (Scale bar represents 25μm.) G) Scanning electron microscopy images of DWJM. One surface appears flat with compact matrix (left lower image) while, less dense tissue with open spaces is identified in other areas (lower right and middle images). (Scale bar for the full picture is 600 μm.) H) Transmission electron microscopy images of DWJM. More electron-dense areas of DWJM (left upper image) and less electron dense areas (right upper image) are observed. No intact cells were observed in any of the panels. (Scale bar for left upper image is 2 μm, for right upper image 10 μm, and for the two lower images 500 nm.).

3.1.1 DNA quantification studies

DNA was isolated from the native WJ matrix and from DWJM and quantified as described above. Mean dsDNA content per DWJM wet weight sample was 1.7 x 10−3 μg/mg (range: 1.4 x 10–3–2 x 10−3 μg/mg), while the mean dsDNA per WJ matrix wet weight sample was 5.1 x 102 μg/mg (range: 3.17 x 10–2–7.33 x 10−2 μg/mg) (Fig 2A). Therefore, 96.6% ± 0.4% reduction in dsDNA was achieved for all the scaffolds analyzed, thus indicating that the majority of cells and nuclei of human umbilical cord were removed.

Fig 2

Quantification of DWJM.

A) DNA quantification study performed on the matrix before decellularization and after decellularization. DWJM showed significantly less DNA compared to the native WJ matrix before decellularization. B) Glycosaminoglycan content assessment of the matrix before and after decellularization. (* Indicates statistical significance (p < .05)).

3.1.2. Protein content analysis

Mass spectrometry revealed that the DWJM matrix pieces were composed of several structural proteins, including collagen I, III, VI, and XII. Transforming growth factor beta (TGFB) was also observed in addition to matrix proteins such as fibronectin-I, which binds to extracellular matrix components for instance collagen, heparin sulfate, tenascin and lumican. A full list of the proteins identified on mass spectrometry evaluation of DWJM is as shown in Table 2.

Table 2

Proteins identified in Decellularized Wharton’s Jelly Matrix (DWJM) by mass Spectrometry.

Protein name	Accession number (1)*	Sequence coverage	MW [kDa]	Theoretical. pI	Peptides number	Unique Peptides
Collagen alpha-3(VI)	219521324	13.70	278.0	8.15	18	18
Collagen type I alpha-1	110349772	6.01	138.8	5.80	7	2
Collagen type I alpha 1	180392	9.13	98.5	6.83	7	2
Collagen, type VI, alpha 1	119629727	12.06	108.5	5.43	8	8
Human Serum Albumin	55669910	16.78	65.2	5.80	7	7
Collagen type I alpha 2	825646	6.49	72.2	7.96	4	4
Collagen type VI alpha-2 isoform 2C2	115527062	13.74	108.5	6.21	8	8
Fibronectin 1	219518912	5.38	239.5	5.88	5	5
G-gamma-hemoglobin	183851	31.68	11.0	6.68	2	2
Protein kinase, DNA-activated, catalytic polypeptide	119607089	0.47	458.5	7.08	1	1
Tenascin C	156229767	4.93	210.4	4.98	4	4
TGFBI, beta-induced transforming growth factor	221044656	19.25	55.7	6.84	4	4
Lumican	4505047	15.68	38.4	6.61	3	3
Collagen, type III, alpha 1	119631314	2.66	106.3	8.10	2	2
Osteoglycin	55957237	13.06	30.4	8.34	3	3
TGFBI beta-induced transforming growth factor	37589544	3.00	75.1	7.23	1	1
Actin, alpha	119612724	12.50	30.3	5.00	2	1
Beta actin, gamma 1	194375299	10.21	37.3	5.71	2	1
HCG2044004 Human chorionic growth hormone	119628289	46.88	3.6	9.32	1	1
Collagen, type XII, alpha 1, isoform CRA_c	119569135	2.22	333.0	5.53	3	3
Hemoglobin alpha 2	13958153	59.21	8.4	7.14	2	2
Immunoglobulin heavy chain variable region	145911949	33.33	9.6	6.52	1	1
Ig G1 H Nie	229601	3.57	49.2	8.54	1	1
Decorin	119617856	14.29	28.0	8.13	2	2
Unnamed protein product	40036688	17.72	17.8	8.38	1	1
N6AMT2 Lysine N-methyl transferase	119628685	29.07	9.8	4.36	1	1
Dynein, axonemal, heavy chain 14	220732359	5.31	40.7	5.21	1	1
Chain D, Crystal Structure Of A Sparc-Collagen Complex	215261061	36.36	3.0	11.00	1	1
Golgin subfamily A member 3 (GOLGA3) protein	38174254	4.63	93.0	5.05	1	1
Glyceraldehyde 3-phosphate dehydrogenase	134254708	14.46	17.3	8.60	1	1
Triacylglycerol lipase (EC 3.1.1.3), hormone-sensitive—human	1082874	3.18	85.4	7.77	1	1
PLEKHG3 protein Pleckstrin homology domain family G	120537866	3.32	80.8	5.40	1	1
OPK V Other protein kinase group, NimA family	38502049	4.01	67.9	8.98	1	1
Plexin D1, isoform CRA_c	119599646	1.09	193.4	6.96	1	1
CDH24 Cadherin 24	28375477	10.79	26.3	5.43	1	1
Beta IV spectrin isoform sigma3	11602888	1.76	148.5	6.37	1	1
FBLN1 Fibulin-1	22761800	3.61	70.5	5.91	1	1
Unnamed protein product	40035675	3.16	68.9	9.32	1	1
Immunoglobulin heavy chain variable region	13171510	52.73	6.2	8.76	1	1
Periostin isoform thy8	166343771	3.19	80.3	8.19	1	1
Transferrin receptor protein 2	33589848	3.37	88.7	6.11	1	1
H2AFJ histone	194382012	17.12	12.1	10.40	1	1
Large tumor suppressor, homolog 2 variant	62089380	1.95	101.4	9.22	1	1
Truncated beta-globin	58201131	47.50	4.5	9.47	1	1
Dermatopontin	27151769	39.8	24	4.82	4	4
Serum albumin preproprotein [Homo sapiens]	4502027	36.29	69.3	6.28	17	17
Ig kappa chain C region	125145	32.08	11.6	5.87	2	2
Fibrinogen beta chain	399492	26.68	55.9	8.27	6	6
Fibrillin-1	311033452	17.69	312	4.93	25	25
Apolipoprotein A-I isoform X2 [Homo sapiens]	530398069	15.73	30.8	5.76	3	3
Ig gamma-1 chain C region	121039	15.5	36.1	8.19	3	3
Mimecan isoform X2 [Homo sapiens]	530391203	11.74	33.9	5.63	2	2
Fibrinogen gamma chain	20178280	9.05	51.5	5.6	2	2
Keratin, type I cytoskeletal 9	239938886	8.35	62	5.24	2	2
Fibronectin isoform 6 preproprotein [Homo sapiens]	47132549	6.8	239.5	5.88	9	9
Fibrinogen alpha chain isoform alpha pre-protein	11761629	6.52	69.7	8.06	3	3
Alpha-fetoprotein	120042	6.4	68.6	5.68	2	2
Keratin, type II cytoskeletal 1	238054406	5.59	66	8.12	3	3
Versican core protein	2506816	2.06	372.6	4.51	3	3
Fibrillin-2	238054385	1.03	314.6	4.86	2	2

* Accession number refers to the accession number in the National Center for Biotechnology Information (NCBI) protein database.

3.1.3. Glycosaminoglycan content analysis

Glycoaminoglycans (GAGs) are glycoproteins with a protein core and long unbranched polysaccharide with repeating disaccharide units. Among other functions, through its carboxylic and /or sulfate ester groups, GAGs can form bridges and link collagens constructing an interconnected network of extracellular matrix to maintain and define shape of connective tissues and organs [17, 18]. Glycosaminoglycan analysis indicated that DWJM contained sulfated GAGs (mean = 0.661 ± 0.107 μg/mg), which was significantly less than that for native umbilical cord Wharton’s jelly tissue (3.0 ± 0.355 ug/mg,