**1. Introduction**

Tissue repair is frequently necessary after skeletal diseases, congenital abnormalities, infections, trauma and surgical procedures after hematological, breast and ovary cancers. Fractures with bone loss often require grafts or implants. Autologous and allogeneic grafts represent about 90% of bone tissue transplants while inorganic matrices represent the other 10% [1,2]. Ideal implants must act as scaffold for bone regeneration with host tissue integration.

Main function of scaffolds is to offer structure and support for migration and specialization of different cells involved in healing. This structure should allow cell adhesion, attachment, differentiation, proliferation and biologic function for repair of the injured tissue [3].

Mesenchymal stem cells (MSCs) are used in tissue engineering [4–6] as an excellent alternative for bone repair since they are able to differentiate in osteoblasts as also in chondrocytes, myocytes, adipocytes and fibroblasts [7]. MSC applied in tissue repair has evolved progressively to improve or even substitute the healing capacity of bone tissue in partial or complete failure of the repair mechanism [8,9].

Combination of live cells with synthetic or natural sca ffolds has been used to produce live tridimensional tissues that are functional, structural and mechanically identical to the original [10–12]. Di fferent compounds have been used as sca ffolds for MSCs [13] and can be classified as synthetic (i.e., hydroxyapatite and calcium triphosphate) [14] or biologic as fibrin biopolymers [15,16].

Synthetic osteoconductive implants have porous structures that promotes bone growth, however, the absence of an osteoinductive potential is still a limitation [17]. Fibrin matrix possesses some special characteristics that make it the sca ffold of choice in tissue engineering [18]. Commercially available fibrin biopolymers are used in di fferent surgical fields as hemostatic agents, healing promoters, cavity sealers and drug delivery in surgical sites [19,20]. Fibrin biopolymers have showed in vitro similar structure and mechanical properties to those of the fibrin clot in vivo [21,22].

Biocompatibility, biodegradability and the capacity to interact with MSC sugges<sup>t</sup> that fibrin biopolymers are important vehicles for cell transplantation [20,21,23]. However, they are derived from human thrombin and fibrinogen that has a risk of infectious disease transmission and limited use due to possible lack of the main components [24–26].

Fibrin biopolymers commercially available today are produced from human thrombin and fibrinogen, being expensive and used only in specific surgical cases. Hence, this study evaluated a new fibrin biopolymer (FBP) composed of a mixture of a serine protease with thrombin-like enzyme activity, purified from *Crotalus durissus terrificus* snake venom and bu ffalo cryoprecipitate as a source of fibrinogen [27].

This new FBP has been used in experimental biomedical applications [28–33] such as nervous tissue [34,35] and bone repair [36] as also on the treatment of chronic venous ulcers in human patients [32,35]. In addition, the FBP enabled in vitro MSC adhesion, growth, had no negative e ffect on cell di fferentiation, and also maintained cell viability [15].

Although many associations of sca ffolds and MSCs are being studied for bone defect healing there are still challenges to be faced [37–40]. Aiming to overcome current method limitations we evaluated the e ffect of this new FBP with MSCs and osteogenic di fferentiated MSCs on the treatment of critical-size defects in rats.

### **2. Material and Methods**

### *2.1. Animals and Ethical Approval*

All experiments were performed in 2-month-old male Wistar rats (*n* = 27) weighing between 200 and 250 g. Animals were housed in polycarbonate cages (4 per cage) and were kept at 21 ± 2 ◦C under a 12-h light/dark cycle and a humidity of 60% ± 10%. The animals had ad libitum access to food pellets of standard rodent diet and water. The Experimental ethics committee for the protection of experimental animal welfare of Botucatu Medical School, Sao Paulo State University, Brazil has approved this study (No. 968-12). The guidelines of the European convention for the protection of vertebrate animals used for experimental purposes and, the Guide for the care and use of laboratory animals and good laboratory practices were fully adopted.

### *2.2. Fibrin Biopolymer (FBP)*

The FBP was kindly provided by center for the Study of Venoms and Venomous Animals (CEVAP), Brazil. Components were distributed in three vials containing thrombin-like enzyme, animal cryoprecipitate and diluent and were kept frozen at −20 ◦C until use [35,41–44]. At time of surgery, contents were immediately mixed according to the manufacturer's package insert.

### *2.3. Cell Isolation and Culture*

Twelve 10-day-old Wistar rats were euthanized with halothane overdose (MAC > 5%) and used as bone marrow donors. Stem cells were harvested by washing of femur marrow cavity with the injection of Dulbecco's modified Eagle's medium (DMEM) (Gibco Laboratories, Grand Island, NE, USA).

The material was pooled, centrifuged at 2000 rpm for 10 min and resuspended in complete culture medium composed of DMEM (Gibco Laboratories) supplemented with 20% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 100 μg/mL of penicillin/streptomycin solution (Gibco Laboratories) and 3 μg/mL of amphotericin B (Gibco Laboratories).

Cells were seeded in 75 cm<sup>2</sup> culture flasks and placed in a 5% CO2 incubator at 37.5 ◦C. Culture medium was changed every 3 days and cell growth and adherence were monitored by inverted microscopy. Cells were subcultured when reached 80% confluence. All experiments were performed with MSCs at passage 3 (P3). To perform the passage, culture medium was discarded; the cells were washed with 2 mL of PBS followed by addition of Tryple Select (Gibco Laboratories) for cell trypsinization and the flask was maintained in an incubator oven for 5 min.

These were centrifuged for 10 min at 2000 rpm and resuspended in culture media. Cells were counted and 1 × 10<sup>6</sup> cells/dose were used in association with FBP for the treatment of the bone defect throughout the experiment [35].

Cells were characterized by flow cytometry (FACS Calibur; BD Pharmingen, San Diego, CA, USA) using monoclonal antibodies for specific positive and negative markers (Table 1) [13,14,45,46]. Assays were performed using 2 × 10<sup>5</sup> cells and data were analyzed using the Cell Quest Pro software after acquisition of 20,000 events. Functional characterization was also performed as cells were differentiated in osteogenic, chondrogenic and adipogenic lineages after the third passage [22,36,47].




### *2.4. Osteogenic Di*ff*erentiation of MSCs*

After cell culture had reached 70% confluence, culture medium was replaced by Stem Pro Osteogenesis Differentiation Kit medium (Gibco Life Technologies A10072-01, Carlsbad, CA, USA), composed of 73% osteocyte/chondrocyte differentiation basal medium (Gibco Life Technologies A10069-01, Carlsbad, CA, USA), 5% osteogenesis supplement (Gibco Life Technologies A10066-01, Carlsbad, CA, USA), 1% penicillin/streptomycin, 1% amphotericin B and 20% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). The differentiation medium was replaced every 3 days for 12 days.

Then, cells were fixed in ice-cold 70% ethanol, washed in distilled water and stained in 2 mL of alizarin red (Invitrogen Life Science Technologies, Carlsbad, CA, USA) for 30 min at room temperature. After the dye was removed, cells were washed four times in distilled water and observed in an inverted light microscope [17,48].

### *2.5. Animals and Surgical Protocols*

Animals were weighed and anesthetized with ketamine solution (1 mL/kg) and xylazine hydrochloride (0.25 mL/kg) intraperitoneally. Cross sections of the thigh through the upper- and middle-third of the femur allowed a critical defect of 5 mm to be performed on the distal epiphysis of the right femur with a low rotation drill (Beltec) under constant irrigation of 0.9% sterile saline to prevent overheating [49]. Postoperative analgesia with intramuscular flunixin-meglumine (1 mg/kg) was performed every 24 h for three days.

Animals were distributed in three experimental groups of 9 animals each: (FBP), the animals were treated with fibrin biopolymer only; (FBP + MSCs) treated with fibrin biopolymer in association with mesenchymal stem cells; and (FBP + MSC-D) treated with fibrin biopolymer in association with di fferentiated mesenchymal stem cells.

Three untreated animals were used as control to assess critical defects throughout the experimental period and evaluated radiographically at 42 dpi.

Cells were mixed in 100 μL of FBP immediately before injection at 1 × 10<sup>6</sup> cells/dose for FBP + MSCs and FBP + MSC-D groups. Surgeries were carried out under sterile conditions.
