Introduction

Bone tissue has a great capacity of self-re- generation; however, complicated injuries such as fractures, or surgically induced le- sions after tumor resection or osteomyelitis are common, in which normal bone healing procedure is disrupted (Zhao et al., 2010). Tissue engineering has recently been devel- oped as a treatment procedure for regener- ation of injured or compromised bone tis- sue. Bone regeneration has involved the use of scaffolds, growth factors and stem cells (Westhrin, Xie, Older, Sikorski, & Strand, 2015). Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can differ- entiate into a variety of connective tissue phenotypes such as osteoblasts, chondro- cytes and adipocytes (Westhrin et al., 2015). One of the most intriguing points in regen- erative medicine, is how to conduct stem cell differentiation toward a specific favorite lin- eage. In vivo, suitable differentiation, prolif- eration and maintenance of potency of stem cells are adjusted by their 3- dimensional microenvironments. Biomaterials can mimic this microenvironment, impressing the in vi- tro differentiation. As a consequence, tissue regeneration research has been principally focused on finding out how differentiation factors (e.g., chemicals, growth factor, extra- cellular matrix components, cell morpholo- gy, matrix stiffness and mechanical forces) play a part in the stem cell microenvironment (Laurent & Mashinchian, 2015).  Among the microenvironmental factors that regu- late stem cell fate and function, mechanical factors have been introduced as important determinants. Mechanical factors including rigidity and topology of the extracellular ma- trix (ECM) can change the fate of stem cells because they  can  sense  physical  features of  their  environment  through  a mechanism

named mechanotransduction (Macqueen, Sun, & Simmons, 2013). A substrate mim- icking surface topography and 3D shape of the original extracellular matrix can be used as a perfect culture substrate for guiding stem cell differentiation. In this regard, several re- searches have been carried out on stem cell differentiation by a cell-imprinting substrate, in which the footprint of the adult differenti- ated cells has been used. Further research has studied the differentiation of stem cells to- ward keratinocytes, tenocytes and chondro- cytes, by the cell- imprinting method, which has suggested a new approach to mechanical stem cell differentiation (Mahmoudi et al., 2013)Kamguyan et al.,  2018)(Mashinchian et al., 2014).

The aim of the current study was to evalu- ate adipose-derived stem cells (ADSCs) dif- ferentiation toward osteogenic linage based on the cell-imprinting method, in vitro and  in vivo.

Materials and Methods

Isolation and Culture of  Adipose-De- rived  Stem  Cells  (ADSCs):   Subcutane- ous adipose tissue samples were surgically harvested from inguinal region of healthy male 250-gram weight, Wistar rats. Tissue samples were washed 3 times in PBS/pen- icillin-streptomycin (300 IU/mL) solution. Then samples were cut into 1×1 mm pieces and digested in 0.1 mg/mL collagenase type II (Sigma, United States) for 1h in an incu- bator. After enzymatic digestion, the solu- tion was filtered through a 150-micrometer filter to remove undigested tissue, followed by neutralization of the enzyme with Dul- becco’s modified Eagle’s medium (DMEM; Gibco, Scotland) containing 10% FBS, then suspension was centrifuged at 1300 rpm   for

5 min and the cell pellets were separated. The solution containing suspended ADSCs was then moved to the culture medium con- sisting of DMEM supplemented with 10% FBS and penicillin–streptomycin (100 IU/ mL, Sigma, United States) in a humidified incubator (37 ºC, 5% CO2). The culture me- dium was changed every three days until a cell confluence of ≥80%.

Isolation and Culture  of Osteoblast: Two  3 day-old neonatal rats were euthanized and sterilized with 70% ethanol. After removing the skin and brain tissue from the skull us- ing a scalpel and tweezers, any excess tissue and cartilage from around the calvaria were scraped off. Similarly,  the  skin,  muscles and soft tissue around the long bones were scraped away. Epiphyses were cut off and then the bone fragments were placed into a tube containing PBS with antibiotics. Bone samples were washed 3 times and chopped into 1mm pieces. Bone samples were incu- bated in 0.25% trypsin for 10 min at 37 ºC. Trypsin solution was discarded and bone fragments were washed in DMEM. After that, samples were incubated in 0.2% colla- genase type II solution for 60 min at 37 ºC. The final solution was kept in a 15-ml tube and centrifuged at 1500 rpm for 5 min. The supernatant was discarded and cell pellets within culture medium were transferred to 6-well cell culture plates. Plates were incu- bated at 37 ºC 5% CO2, until the cells be- came confluent after  5 days  (Taylor,  Shah, & Orriss, 2014). Osteoblast characterization was performed by alkaline phosphatase mea- surements and alizarin red staining (Rosa, 2005)(Nikukar et al., 2013).

Fabrication of Cell-Imprinted Substrates: Polydimethylsiloxane (PDMS, SYLGARD 184, RTV, Dow  Corning,  United  States) was employed to fabricate the cell-   imprint-

ed substrates. The silicone resin and curing agent were combined with a 10:1 ratio. Con- fluent osteoblast plates which were fixed by 4% glutaraldehyde solution previously were used as template for imprinting process. The resin-curing agent mixture was poured very gently onto plates and kept at 37 °C for 48

h. The patterned PDMS was peeled off the osteoblasts, followed by several washing steps using 1M NaOH solution to eliminate remained cells and debris. These prepared substrates were fixed in the bottom of cell culture polystyrene plates and after steriliz- ing procedure, substrates were made ready for stem cell seeding.(Allan et al., 2018) (Bonakdar et al., 2016)

Mesenchymal Stem Cells Seeding on Substrate: ADSCs which were previously isolated and cultured, were transferred onto osteoblast-imprinted substrates, by the ratio of 1×104 cells per each 3-cm culture plate. Culture medium for  this  step  was  similar  to that used in ADSCs culture. Plates were maintained in a humidified incubator (37 ºC, 5% CO2) up to cells covering all of the pat- terned PDMS surface (14 days).

Characterization of Stem Cells  Cultured on PDMS Substrates: Alizarin Red staining and Alkaline Phosphatase (ALP) measure- ment was performed in order to determine osteogenic properties and compare with un- differentiated ADSCs. The cells in two plates were fixed by 4% glutaraldehyde  solution for 24h and washing with PBS, staining with alizarin red stain was done for 45 min, then they were washed with PBS for removing residual stain. Red plaques were seen by an inverted optical microscope (Olympus IX71, Japan) and compared with what was ob- served in ADSCs.

Osteogenic differentiation was investi- gated   via  ALP  activity   measurements   in

cell supernatants at days 7 and 14. Collected samples were stored at -20 ºC and tested by an alkaline phosphatase kit (Sigma, United States) according to its standard protocol. The absorbance at a wavelength of 405 nm  in a ELISA reader spectrophotometer was measured. (Thibault et al., 2010)

Cell Implantation in rat calvarial defect: Cells were seeded on a commercial colla- gen scaffold (Nano-Zist-Arayeh, Iran) at the rate of 8×105 cells per each 8 cm diameter round scaffold.

The three groups of study (described as differentiated cells by osteoblast-imprint-  ed substrate, ADSC and cell-free scaffold) were implanted in rat calvarial defects for 4 weeks. The sample size (n = 5) was selected on the basis of previous studies. (Ekaterina et al., 2012)

Twenty male Wistar rats (2 months old; weight = 300–350 g) were  maintained  in  the animal facility for 2 weeks to become adapted to housing conditions. The rats were anesthetized with intramuscular administra- tion of ketamine (80 mg/kg) and xylazine (10 mg/kg) combination. The surgical site was shaved and scrubbed with iodine. Utilizing sterile instruments and aseptic circumstance, a cranial skin incision was made in an an- terior to posterior direction along the mid- line. Soft tissue, musculature and periosteum were dissected and reflected to expose the calvaria. Central full-thickness critical size defect 8 mm in diameter was created using   a saline-cooled trephine drill. The dura mater was not disturbed. Then defect was implant- ed with the scaffold, the periosteum and skin were reclosed by suture. Post-surgery, the an- imals were injected with a dose of enroflox- acin subcutaneously. After 4 weeks, the ani- mals were sacrificed by CO2 inhalation, and the  calvarial  defect  sites  with   surrounding

bone were harvested for evaluation (Ekateri- na et al., 2012) (Narayanan et al., 2013).

Histopathologic Investigation: Samples, including the implantation sites with sur- rounding bone, were fixed in 10% buffered formaldehyde for 5 days, then transferred  into decalcifier solution (10% Ethylenedi- aminetetraacetic acid, pH=7.2). After com- plete decalcification (14 days), samples were dehydrated in increasing grade series of eth- anol and embedded in paraffin using routine histological techniques. Then the specimens were sectioned at 5 μm by a rotary micro- tome and stained with hematoxylin and eo- sin (H&E). Transmitted light images of the stained sections were taken with a micro- scope (Nikon Eclips E600) connected to a CCD camera. The sections were analyzed for new bone formation, tissue components, vas- cularization and inflammatory response (HI- ATT, 2007) (Park, Kim, Moon, & Na, 2009).

Results

Alizarin red staining of cells in day 17 rep- resented a great difference between ADSCs and differentiated cells on osteoblast-im- printed substrate. Obvious red foci of secret- ed mineral matrix in differentiated cell group indicate remarkable osteogenic differentia- tion. On the other hand, in ADSCs group, no red stained area was observed (Fig.1).

Statistical analysis of  ALP  measurement in cellular supernatant of days 7 and 14, re- vealed significant differences between two groups, in a way that differentiated cells showed greater amount of ALP than ADSCs in both time points. (p<0.05)

In histopathological evaluation, specimens implanted with differentiated cells showed greater bone regeneration compared to those with ADSCs and scaffold alone. This newly formed bone along the defects in group differ-

Figure 1. Alizarin red staining of cell culture plates. There is a remarkable red colored area in differentiated cells plates. It represented enormous mineral matrix production as an important characteristic of osteogenic lineage. A photomicrograph of staining cell culture plate, shows the mineralized matrix in a greater magnification.

Figure 2. Photomicrographs of calvarial defect area. Pictures A, B and C is related to differentiated group, and D represents part of defect in group ADSCs. Newly formed bone (NB) lamella in the center of defect was seen(A). Within the connective tissue (CT) in differentiated group there is some foci of intramembranous ossification (blue arrowhead in B). Numerous new- ly formed vessels (*) showed higher rate of angiogenesis in differentiated group(C). Remnants of scaffold showed by a white “S” (C). In ADSCs group except thine to moderate connective tissue, there is not seen new bone lamella or great number of new vessels (D). Hematoxylin & Eosin staining. Bar=200 micrometers (A,C) Bar= 100 micrometers (B,D).

entiated cells was observed both in the center of defect and in the edge of defect, adjacent to old bone. While in groups ADSCs and only scaffold no new bone lamella was seen in the center of defects, just limited bone formation from the edges of defect was observed. New- ly formed vessels in differentiated group was more than the other two groups and these structures were distributed all around the defect in spite of ADSCs and scaffold group in which vessels mostly formed near the de- fect edges. In differentiated cell group, thick connective tissue with active spindle-shaped fibroblasts, numerous new vessels  and foci of intramembranous ossification in addition to newly formed bone lamella, connected the edges of defect to each other. On the other hand, in groups ADSCs and scaffold, thin  to

moderate connective tissue with less active fibroblast in comparison with differentiated group, without any sign of ossification, con- nected the defect edges  (Fig. 2).

Although differentiated cell  group  most- ly showed moderate mononuclear cell in- flammatory  response,  in  some   specimens in which the scaffold was not completely degraded, prominent giant cells within the remnant scaffold were  seen.

Discussion

Sheep Bone is capable of repairing lim- ited defects through remodeling. Compli- cated bone lesions (e.g., defects related to severe trauma, tumors, diabetes, osteone- crosis and osteoarthritis), would  be  helped by   cell-based   therapy.   Tissue engineering