Implanted subcutaneous versus intraperitoneal bioscaffold seeded with hepatocyte-like cells: functional evaluation

The X-linked bleeding disorder, hemophilia A, is caused by defective production of factor VIII (FVIII). Hemophilic patients require regular FVIII infusions. Recombinant factor replacement poses the safest line of therapy. However, its main drawbacks are high expenses and the higher liability for formation of inhibitors. Recent studies confirmed the ability of bone marrow-derived stem cells to secrete FVIII. This study aims to generate bioscaffold from decellularized liver and subsequently seed it with trans-differentiated human stem cells into hepatic-like cells. This scaffold can then be implanted intraperitoneally or subcutaneously to provide FVIII. After generation of the bioscaffold, seeding of discoid scaffolds with trans-differentiated human hepatocyte-like cells was performed. Then, the generated organoid was implanted into peritoneal cavity or subcutaneous tissue of experimental rats. Serum human FVIII was significantly increased in rats subjected to subcutaneous implantation compared intraperitoneal implantation. Immunostaining for detecting Cytokeratin 19 and human anti-globulin confirmed the presence of mature human hepatocytes that were significantly increased in subcutaneous implanted scaffold compared to the intraperitoneal one. Implantation of decellularized bioscaffold seeded with trans-differentiated stem cells in rats was successful to establish production of FVIII. Subcutaneous implantation showed higher FVIII levels than intraperitoneal implantation.


Introduction
Hemophilia is an x-linked inherited disease caused by deficiency of clotting factors VIII (Hemophilia A) or IX (Hemophilia B). Hemophilia is associated with recurrent and spontaneous bleeding, mainly internal bleedings into joints or muscles [1]. Hemophilia A occurs in 1 out of 10,000 male births, whereas hemophilia B occurs in 1 out of 30,000 male births. The prevalence of hemophilia A changes with the reporting country, ranging from 5.4 to 14.5 cases per 100,000 male individuals [2].
Management of Hemophilia A is directed mainly toward symptomatic prevention of bleeding through replacement of defective factor. Several clotting factor Open Access *Correspondence: Asmaa82shamseldeen@gmail.com; Asmaa. abdulwahab@kasralainy.edu.eg 3 Physiology Department, Faculty of Medicine, Cairo University, Giza, Egypt Full list of author information is available at the end of the article VIII concentrates are available for managing hemophilia A. Biotechnology is able to develop recombinant factor concentrates for replacement therapy [3].
However, the disadvantages of replacement therapy are its high cost, life-long dependence as well as antibody production [4]. The most serious complication of replacement therapy in hemophilia is antibody production [5]. These antibodies are polyclonal high-affinity immunoglobulin G directed against the factor VIII protein leading to an increase in the management cost, morbidity and mortality [6].
Increasing circulating level of factor VIII by 1-5% could substantially reduce bleeding tending. Being the source of plasma factor VIII, liver cells became the main hope for treating hemophilia A [4]. Moreover, to find a feasible long-life line of treatment such as liver cell based therapy emphasizes a major concern. Recently, Rossi and colleagues have reported improvement viability and functions of transplanted hepatocytes seeded onto naturally developed bioscaffold by decellularization process [7].
The formed bioscaffold is composed of hepatic-extracellular matrix (ECM) such as collagen, elastin and proteoglycans, and no remaining functioning cells, but still rich in factors that could promote cellular interaction, growth and survival [8]. Thus, seeding the decellularized liver bioscaffold with stem cells can enhance their growth, therapeutic efficacy and differentiation into functioning cells [7] Mesenchymal stem cells (MSCs) represent a feasible cell source for cell therapy for their accessibility, expansion potential, high regenerative capacity, and safety in a large number of clinical trials [9]. Previous studies revealed that MSC-derived from different sources (both mice and humans) can be differentiated into hepatocytes, using various protocols and techniques in vitro [10].
Notably, this study was designed to evaluate the function and histology of decellularized liver bioscaffold seeded with MSC-derived hepatocyte-like cells implanted in rats and compare between subcutaneous and intraperitoneal routes, finally to verify the ability of both scaffolds to survive, function and secrete functional proteins.

Generation of bioscaffold
Surgical procedure: Animals were euthanized, then a longitudinal anterior abdominal incision was made. A 5-kg piglet liver was removed surgically following intraoperative ligation of the hepatic artery and splenic vein to avoid leakage of the detergents used in decellularization process. The liver was dissected with the capsule intact.

Decellularization process
The liver was dissected and the portal vein was cannulated and connected to pump that perfused the liver with sterile distilled water. 15 L of distilled water was injected in the liver until all blood inside the liver was washed. Subsequently, 6 L of detergent were injected (1% SDS + 0.1% ammonium hydroxide, Sigma Aldrich, St. Louis, USA) to kill the hepatocytes and the endothelial cells. Decellularized liver bioscaffolds were detected by being transparent then confirmed laboratory by the absence of positive staining for hematoxylin [11] of liver sample.

Mononuclear cell separation from human bone marrow samples
The experiments involving humans were carried out in accordance with The Code of Ethics of the World Medical Association (Human Declaration of Helsinki). After institutional ethical committee approval and taking informed consent from the participants, three-milliliter of bone marrow was obtained by aspiration from the posterior iliac spine. The mononuclear cells were obtained from fresh human bone marrow samples after using Ficoll-Hypaque density gradient (density, 1.077; Biochrom KG, Berlin) [12]. Cell pellets were suspended in 1 mL serumfree DMEM media and counted using a hemocytometer. Counting and viability was assessed by the vital stain trypan blue (0.4%) exclusive dye (Sigma).

Mesenchymal stem cell (MSCs) separation and identification
After isolation, primary culture and subculture of MSCs were done according to Soleimani and Nadir technique [13]. Flasks used were incubated in a horizontal position in humidified incubator at 37 °C and 5% CO 2 . The first change of media was accomplished at the third to the fifth day to remove non adherent cells, and then the media was changed twice weekly until reaching 70-90% confluency which was assessed by the inverted microscope. The adherent cells were harvested by trypsinization according to Bieback method [14]. After trypsnization, cells were examined under the microscope, counted and the viability was assessed using the trypan blue dye. The cells released from primary culture were divided into two parts, one used for MSCs identification using flow cytometry and the other part for subculture and induction of hepatocyte differentiation. Flow cytometry was done and results were declared as percentage of cells expressing CD 105 + , CD 73 + and CD 271 + and negative for CD 45 + and CD 34 + in the gated population of mononuclear cells (Fig. 1).

Induction of hepatocyte differentiation
Induction of hepatocyte differentiation was done according to Li method [15]. At this step, 14 samples were incubated with hepatocyte growth factor (HGF) 20 ng/mL together with fibroblast growth factor (FGF) 10 ng/mL for induction of differentiation, whereas another six samples were used as a control.

Evaluation of hepatocyte differentiation
Evaluation of hepatocyte differentiation was done using several measures firstly under inverted microscope, and dishes were examined for morphological changes denoting differentiation.

Gene expression of AFP and albumin was detected using real time: PCR
The total RNA was extracted from hepatic-like cells according to the manufacture instructions. In brief, the first denaturation step was done by incubating reaction tubes for 10 min at 94 °C, then followed by 40 cycles of 1 min at 94 °C (denaturation). For AFP and albumin, 1 min was applied for 58 °C and 56 °C for β-actin (annealing) and 1 min at 72 °C (extension) and the final elongation step was at 72 °C for 7 min. The primer sequence for AFP is sense; ACC ATG AAG TGG GTG GAA TC, and antisense; TGG TAG CCA GGT CAG CTA AA, for albumin is sense; GCA TCC TGA TTA CTC TGT CG, and antisense; GTT CAC CAA GGA TTC TGT GC, and for β-actin is sense; GGG CAT GGG TCA GAA GGA TT, and antisense; GAG GCG TAC AGG GAT AGC AC.
On day 14, the media were condensed five times by freeze drying before estimating concentrations of albumin and AFP [16]. Albumin and AFP were also measured in the samples using radioimmunoassay method. AFP was measured using AFP monoclonal antibody (P5B8) a product of ThermoFisher Cat #MA5-14666, and albumin was measured using human serum albumin monoclonal antibody (1E1) a product of TheimoFisher Cat # MIH3001.

Seeding the formed scaffolds with differentiated MSCs
The differentiated MSCs were resuspended at a concentration of 2 × 10 6 /50 μL/cubic scaffold. Using a 0.5 mL insulin syringe, cells were drawn up then released drop by drop over scaffolds to cover the prepared decellularized tissue.

Animal experimental design
All animal experiments complied with the ARRIVE guidelines and carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. This study was conducted on 21 adult male albino rats with average body weight 200 g, purchased from and housed in the Animal House, Faculty of Medicine, Cairo University, and experimental procedures were done in Physiology laboratory, Faculty of medicine according to the guidelines for the care and use of experimental animals of Cairo University and were divided into the following three groups: Group I (control): It included seven rats that were left without any intervention. Group II (subcutaneous group): It included seven rats that were implanted subcutaneously with the generated bioscaffold after differentiation in culture for14 days. Group III (Intraperitoneal group): It included seven rats that were implanted intraperitoneally with the generated bioscaffold after differentiation in culture for 14 days.

Laboratory investigation
By the end of the study (after 10 days), blood samples were collected and plasma was separated for detecting level of FVIII using human FVIII ELISA kit (total FVIII antigen) (ab272771). The detection method was colorimetric. Rats of all groups were sacrificed after 10 days. The rats were sacrificed by cervical dislocation. Skin and peritoneal specimens were fixed in 10% formol saline for 24 h. Paraffin blocks were prepared and 5-μm-thick serial sections were subjected to the following studies in the Histology Department, Faculty of Medicine, Cairo University:

Histological study
After preparation of paraffin blocks, serial sections from all studied groups were stained with;

Morphometric study
Computer-assisted image analysis was performed using Olympus camera connected to Olympus microscope, using interactive measurements menu. The assessment of area percent (%) of AFP, CK19 (in both culture tissue and experimental groups) &HAG was measured using binary mode. The measurements were done within 10 non-overlapping fields of each specimen using × 100 magnification.

Statistical methods
Statistical analysis and calculations were performed using Statistical Package for the Social Sciences (SPSS) version 16. The comparison between the different groups was analyzed using ANOVA test, followed by Bonferroni post hoc test to detect which pairs of groups caused the significant difference. P values < 0.05 were considered statistically significant.

Bioscaffold quality control and characterization
Enhanced production of human factor VIII with implantation of bioscaffolds: Therapeutic potential of implanted bioscaffolds documented production of human factor VIII in group II and III. However, serum level of HF VIII was significantly increased in rats subjected to subcutaneous compared to animals received bioscaffold by intraperitoneal implantation (Fig. 2).

Cell morphology presenting differentiation of MSCs
Differentiation of human mesenchymal stem cells to hepatic phenotype was confirmed by inverted microscope (Fig. 3).

Culture results
Examinations of cellular differentiation for detection of differentiated hepatocyte-like cells of the culture scaffold Fig. 2 Estimated serum levels of human factor VIII in rats subjected to subcutaneous and intraperitoneal implantation of bioscaffold. Data presented as SD. *Statistically significant compared to the group I (P < 0.001). # Statistically significant compared to the group II (P < 0.001) was done using H&E and immunohistochemical stains for both alpha-fetoprotein (AFP) and cytokeratin 19 (CK19). Results showed on day 0 no differentiated cells detected, on day 7 increased numbers of differentiated cells and on day 14 the cells increased in H&E and in CK19 while decreased in AFP immunostaining (Fig. 4).

Estimation of albumin and AFP secretion in culture media
Data confirmed successful induction of differentiation of human bone marrow mesenchymal stem cells (HBMSCs). That was reflected on significant increase in mean value of both albumin (1.06 ± 0.03 µg/mL) and AFP (20.8 ± 0.94 µg/mL) in culture media of differentiated stem cells compared to the mean values of albumin (0.2 ± 0.01 µg/mL) and in AFP (12.93 ± 0.62 µg/mL) estimated in culture media of the control undifferentiated cells.

Histological results
Examination of skin sections from Group I (control group) showed in H&E-stained sections normal epidermal and dermal layer with normal hair follicles and thin keratin layer while showed in immunostained sections −ve skin reaction. Examinations of skin sections from Group II (subcutaneous group) showed in H&Estained sections hepatocyte-like cells at the bases of the hair follicles. In sections, stained with AFP showed +ve immunostaining cells detected at the roots of hair follicles. While in sections stained with CK19 showed +ve immunostaining cells detected at the shafts of hair follicles (Fig. 5). Examinations of intraperitoneal sections from Group I (control group) showed in H&E-stained sections normal intraperitoneal tissue with some adipose cells and some muscle fibers within loose CT layer while in immunostained sections showed −ve intraperitoneal reactions. Examinations of H&E-stained sections from Group III (intraperitoneal group) showed hepatocytes-like cells at the scaffold with the dilated blood vessel. In both sections stained with AFP and with CK19 showed +ve immunostaining cells detected at the scaffold (Fig. 6). Examinations of sections stained with HAG showed in Group II (subcutaneous group) +ve immunostained cells detected at the shaft of hair follicle and Group III (intraperitoneal group) showed +ve cells detected at the scaffold and blood vessels (Fig. 7).

Morphometric results
(a) On day 14, culture tissue showed a significant decrease in the mean area% of AFP immune positive cells and a significant increase in the mean area% of CK19 immuno +ve cells as compared to day 7 (Table 1). (b) Mean area% of AFP +ve cells, area% of CK19 and area% of HAG +ve cells immune-expression in control and experimental groups showed statistically significant increased compared to the group I. Group II showed a significant decrease in the mean area% of AFP and a significant increase in area% of CK19 and in area% of HAG +ve cells as compared to group III (Table 2).

Enhanced production of human factor VIII with implantation of bioscaffolds
Therapeutic potential of implanted bioscaffolds documented production of human factor VIII in group II and III. However, serum level of HF VIII was significantly increased in rats subjected to subcutaneous compared to animals received bioscaffold by intraperitoneal implantation (Fig. 2).

Discussion
The biologically developed scaffolds from animal organs by the process of decellularization can be used to generate a transplantable fully functioning organ [17]. In the present work, we used decellularized pig liver as a bioscaffold for transplantation of hepatocytelike cells (HLCs) differentiated from human bone marrow mesenchymal stem cells (MSCs). The concept of using decellularized scaffolds was explained by Uygun et al. [17] who stated, these scaffolds can provide a 3D environment helping cell attachment, proliferation, and promotes its function. In our work, the transplanted HLCs were capable of achieving their function in vivo by secreting human factor VIII that was detected in serum samples of the experimental rats. It was reported that human MSCs from bone marrow, umbilical cord blood, adipose tissues, and fetal bone marrow can differentiate to HLCs [18]. The benefits of MSCs were clarified by Croce et al. [19] who mentioned that MSCs are known by simple isolation, high proliferation abilities and can differentiate into HLCs under proper culture conditions in vitro.
Hepatocyte-like cells derived from bone marrow MSCs have the potential to overcome the limitations of primary human hepatocytes for clinical application in treating liver diseases [20] such as the quality and variability of their metabolic/functional activity. Even more, hepatocytes can't be easily maintained in culture for extended time [21].
Growth factors can selectively alter cell function and promote MSC proliferation, migration, and differentiation. Accordingly, researchers have tested different protocols to differentiate these cells into other cell types such as hepatocytes. Different growth factors and cytokines are used for sequential differentiation such as FGF, HGF, oncostatin M, and dexamethasone [10].
In the present study, HBMSCs were isolated, cultured and then differentiated into HLCs. On day 14 of the culture, the differentiated cells showed a significant increase in the level of AFP and albumin in the culture media as compared to the control (undifferentiated cells).These results were in accordance to previous Study which confirmed hepatic differentiation by AFP and albumin level [22].
During differentiation, the morphology of HBMSCs changed and the cells expressed hepatocyte-specific antigens as AFP (immature marker) which increased on day 7 and decreased gradually on day 14, on contrary CK19 (mature marker) decreased on day 7 and increased gradually on day 14.These findings agreed with previous work reported that, AFP is an immature marker and albumin synthesis is a mature marker for hepatocytes [23]. Improvement of liver cyto-architecture is indicted by increasing the expression of albumin and reduced the level of AFP [24]. This is consistent with the fact that AFP is expressed in endoderm and in fetal hepatocytes and that after birth the AFP gene is selectively silenced [25]. CK19 is a marker for hepatic oval cell which is a bipotent progenitor cell that can differentiate into both mature hepatocytes and bile duct epithelial cells [26].
In this study, the clusters of HLCs in the decellularized liver scaffold were implanted subcutaneously (group II) and intraperitoneally (group III). The subcutaneous implantation of the decellularized scaffold presented good histocompatibility [27]. In subcutaneous implantation, there are a few concerns regarding implantation such as degradation and inflammation [28].
Implantation of biomaterials stimulates the host immune reaction and enhances recruitment of macrophages that could be attracted by the local bulk of pro-inflammatory cytokines and growth factors allowing   [29]. In addition, other studies demonstrated stimulation of inflammatory and immune responses after implantation of acellular scaffolds in animal models. The decellularization approaches induce elimination of antigenic parts of the tissues such as the nucleus and other structural elements. However, the extracellular matrix component of the scaffold could stimulate the host immune response, [30,31] causing accumulation of myofibroblasts leading eventually to fibrosis [32]. Moreover, the peritoneal membranes are covered by mesothelial cells that have the ability of mesothelial-tomesenchymal transition, which is a type of extracellular matrix. The latter contributes to development of peritoneal fibrosis upon chronic irritation [32,33]. Thus, it may weaken therapeutic efficacy of intraperitoneal implant.

Conclusion
Decellularization strategy for synthesis of bioscaffold seeded with hepatocyte-like cells has the potential to present an alternative for recombinant factor VIII replacement to treat hemophilia patient. Increased mean values of serum FVIII in groups subjected to subcutaneous implantation make subcutaneous more preferable than intraperitoneal one.  Table 2 Mean area% of AFP +ve cells, area% of CK19andarea% of human antiglobulin +ve cells immunoexpression in control and experimental groups ± SD * Significant increased compared to the group I (P < 0.001). # Significant increased compared to the group II (P < 0.001). ^ Significant decreased compared to the group II (P < 0.0)

Group
Area % of AFP +ve cells Area % of CK19 +ve cells Area % of human antiglobulin +ve cells