The role of TiO2 nanotube surface on osseointegration of titanium implants: Biomechanical and histological study in rats

The nanoscale surface of titanium has been studied to improve the cellular recognition of the biological microenvironment and to increase bone–implant interaction. The aim of this study was to analyze the effect of a titanium oxide (TiO₂) nanotube surface with a machined surface on osseointegration tibia implants without primary stability. This study used an experimental design, divided into two groups (n = 16): commercially pure titanium machined implants (Cp‐Ti Ma) and commercially pure titanium anodized implants (Cp‐Ti An). Titanium nanotubes were produced by anodic oxidation, and the topography of surface was analyzed using field emission scanning microscope (FE‐SEM). The implants (2.1 × 2.8 mm Ø) were surgically placed in the right tibia (defects with milling drill 2.5 × 3.2 mm Ø) of 32 Wistar male rats (250–300 g). The animals were euthanized at 7 weeks postoperatively. The maximum value of removal torque was measured (N/cm) in the right tibia half of each group (8 animals/8 tibiae); the other half of each group underwent a nondecalcified protocol, stained with Stevenel blue/Alizarin red, and the formation of bone tissue in close contact to the implant was measured. The obtained data were analyzed statistically (t test). Differences were considered statistically significant for α < 0.05. Cp‐Ti An implants were significantly higher in removal torque and peri‐implant bone healing compared with Cp‐Ti Ma implants (p < .01). Within the limitations of this study, it was observed that the surface modification of titanium by anodization (TiO₂ nanotubes) can improve osseointegration, and this may be very useful to reduce the time required for peri‐implant bone formation.     

Feasibility of detecting trabecular bone around percutaneous titanium implants in rabbits by in vivo microfocus computed tomography

Objectives: The goal of this study was to examine the feasibility of in vivo imaging of trabecular bone around titanium implants by means of microfocus computed tomography (micro‐CT) and the use of rabbits for this purpose. Materials and Methods: Ten male rabbits type Hollander, received a titanium implant (1.7 mm diameter and 10 mm length) in the trabecular bone of the left tibia. Seven weeks later a micro‐CT scan was taken. Four rabbits were used to monitor potential harmful effects from X‐ray absorption until 4 weeks after scanning. A second group of six rabbits was used for testing the hypothesis that a good correlation exists between in vivo micro‐CT images and histological images of trabecular bone around titanium implants. The six rabbits were scanned and sacrificed immediately. The tibias were extracted and submitted to standard histological procedures. This resulted in a total of 12 histological sections and their corresponding 12 micro‐CT images. Bone area measurements were performed at the left and right side of the implant in three regions: 0–500, 500–1000 and 1000–1500 μm distance from the implant interface. Intra‐class correlations (ICC) were calculated between both techniques. Results: The four rabbits did not show any sign of radiodermatitis 4 weeks after scanning. In the micro‐CT images of the group of six rabbits, trabeculae are visible, but not well defined, due to the presence of noise in the image. The ICC for the right implant side were 0.44 for zone 0–500 μm, 0.48 for zone 500–1000 μm and 0.40 for zone 1000–1500 μm. The ICC for the left implant side could not be calculated. Conclusion: A low agreement was found between the bone measurements from histology and in vivo micro‐CT images. The use of the in vivo micro‐CT for trabecular bone imaging around metallic implants should be restricted to track tendencies in follow‐up studies.     

Design of a stability-detecting device for dental implants

Resonance frequency (RF) analysis technology was used to design a dental implant stability detector. The device uses a miniature-sized electromagnetic triggering rod to elicit vibration in a dental implant. Vibrational signals were recorded via an acoustic receiver. To assess the in vivo performance of the test apparatus, animal models were used. Implants were placed in the left tibia of 12 rabbits using a conventional surgical procedure. Standard 3.2 mm x 8 mm implants were placed in each test tibia with pre-tapping cavities of 3.2 mm and 3.7 mm diameters to simulate either a 'well-fitting' or a 'loosely fitting' situation. The RF values of the test implants were detected by the newly developed device which was directly mounted on the healing abutments of the implants. The results showed that the RF values of the implants under well-fitting conditions significantly increased (p < 0.01) 3 weeks after surgery and reached a plateau at around 6-7 weeks. Meanwhile implants with higher initial RF values had shorter healing times and higher final RF values at the plateau. Based on these findings, it was concluded that the idea of using the current designed device for detecting the degree of bone healing during the osseointegration process seems feasible.     

Bacterial biofilm on successful and failed orthodontic mini‐implants—a scanning electron microscopy study

Mini‐implants have been extensively used in Orthodontics as temporary bone anchorage devices. However, early failure of mini‐implants due to mobility might occur and the colonization of their surfaces by pathogenic bacteria has been referred to as one of the contributing factors. In this study, scanning electron microscopy (SEM) was used to assess the presence of microorganisms adhered to the surface of mini‐implants that failed due to loss of stability. Twelve self‐drilling titanium mini‐implants (1.6 mm diameter × 9.0 mm long) were collected from 12 patients undergoing orthodontic treatment—7 successful and 5 failed mini‐implants. The mean time of permanence in the mouth was 15.8 and 2.4 months for successful and failed mini‐implants, respectively. The devices were placed in the maxilla and/or mandible and removed by the same surgeon and were processed for SEM analysis of the presence of microorganisms on their surfaces (head, transmucosal profile, and body). Extensive bacterial colonization on mini‐implant head and transmucosal profile was observed in all successful and failed mini‐implants. None of the failed mini‐implants exhibited bacteria on its body and only one mini‐implant belonging to the successful (stable) group exhibited bacteria on its body. The results did not suggest a relationship between failure and presence of bacterial colonies on mini‐implant surfaces. Microsc. Res. Tech. 78:1112–1116, 2015. © 2015 Wiley Periodicals, Inc.     

Laser ablation in titanium implants followed by biomimetic hydroxyapatite coating: Histomorphometric study in rabbits

Titanium surface texture and chemistry modification successfully improves the host response and consequently the bone-to-implant contact surrounding dental implants. The aim of the present study was to investigate, using histomorphometrical-analysis, the effects of titanium surface modification by laser-ablation (Nd:YAG) followed by thin chemical deposition of HA. Forty-eight rabbits received one implant by tibiae of AS-machined (MS), laser-modified (LMS), or biomimetic hydroxyapatite-coated (HA) surface. Bone-to-implant contact (BIC) and bone area (BBT) were evaluated after 4, 8, and 12 weeks, at cortical and cancellous regions. Average BIC in the cortical region was higher (P < 0.001) on the LMS and HA implants for all periods, with no differences between LMS and HA. For the cancellous area, the LMS and HA implants showed higher (P < 0.01) BIC than MS at the initial periods. The LMS and HA showed similar values in the cortical region, but a tendency of higher values for HA in the cancellous region was observed in all periods. For the BBT, the differences were found only between HA and MS after 4 weeks in the cortical region (P < 0.05), and after 12 weeks in the cancellous area (P < 0.05). Our results showed that HA biomimetic coating preceded by laser treatment induced the contact osteogenesis and allowed the formation of a more stable bone-implant interface, even in earlier periods.     

Two‐ and three‐dimensional profilometer assessments to determine titanium roughness

In this study, a comparative topography analysis of titanium (Ti) surfaces was performed using two‐ (2D) and three‐dimensional (3D) profilometers. Ti samples were either only sandblasted (SB) using Al₂O₃ particles or were SB and received an additional chemical treatment using a solution of 4% HF (SLA). Samples with no treatment were used as a control group (C). Therefore, three different surfaces were evaluated: SB, SLA and C. The Ti surface topographies were evaluated by scanning electron microscopy. An Ra roughness measurement was performed on each Ti sample by 2D and 3D profilometers. Surface roughness was also characterized using amplitude, spatial and hybrid 3D parameters. 2D and 3D profilometer analyses produced very close results. Mean Ra values range from 0.19 to 0.25 (C, p<0.05), 0.84 to 0.99 (SLA, p<0.05) and 0.98 to 1 µm (SB, p>0.05). The statistically different Ra values depending on the surface studied may be explained by methodological and technical differences. Also, 3D profilometer seems to be the more appropriate analytical method for measuring the roughness of Ti surfaces because it also describes surface organization. SCANNING 31: 174–179, 2009. © 2009 Wiley Periodicals, Inc.     

Fabrication and in vivo evaluation of Ti6Al4V implants with controlled porous structure and complex shape

Electron beam melting process was used to fabricate porous Ti6Al4V implants. The porous structure and surface topography of the implants were characterized by scanning electron microscopy (SEM) and digital microscopy (DM). The results showed that the pore size was around 600 and the porosity approximated to 57%. There was about±50 μm of undulation on implants surfaces. Standard implants and a custom implant coupled with porous sections were designed and fabricated to validate the versatility of the electron beam melting (EBM) technique. After coated with bone-like apatite, samples with fully porous structures were implanted into cranial defects in rabbits to investigate the in vivo performance. The animals were sacrificed at 8 and 12 weeks after implantation. Bone ingrowth into porous structure was examined by histological analysis. The histological sections indicated that a large amount of new bone formation was observed in porous structure. The newly formed bone grew from the calvarial margins toward the center of the bone defect and was in close contact with implant surfaces. The results of the study showed that the EBM produced Ti6Al4V implants with well-controlled porous structure, rough surface topography and bone-like apatite layer are beneficial for bone ingrowth and apposition.     

Osseointegration of hydroxyapatite and remodeling‐resorption of tricalciumphosphate ceramics

Background: Cancellous bone defects surrounded by still intact bone structures never heal. Ceramics offer a solution providing osteoconductive scaffolds. Purpose: The purpose of the study is to evaluate whether structured β‐TCP and HA implants can reconstruct cancellous bone defects, which role micro‐ and macro‐porosity, stiffness and surface area play; finally the indication for both materials based on its resorbability. Material & Methods: 10 German Shepard dogs were operated on both tibial heads implanting shell‐like fully interconnected ceramic cylinders, using a wet grinding hollow drill coated with diamonds. β‐TCP was compared with HA. A polychromatic sequential labelling with 4 different fluorochromes controlled bone formation dynamics. Non‐decalcifying histology after perfusion fixation and vessel casting was performed. μ‐CT was combined with high resolution microradiography and histology on thin ground crossections. The stages after 6 weeks, 2, 3, 4 months and 15 months were evaluated. Results: In spite of osseointegration of HA and β‐TCP, the osseointegration of both materials was completely different. Both shell‐like bone void fillers were osseointegrated in a sandwich‐like manner. HA yielded primarily a reinforcement of the recipient's cancellous‐bone bed and full osseointegration after 4 months, whereas β‐TCP‐implants were fully osseointegrated after 6 weeks. HA did not show signs of resorption. The resorption of the β‐TCP resulted during remodelling. The final stage showed restitution “ad integrum” of the β‐TCP defects with a physiological architecture, whereas HA was integrated in the cancellous bone construction providing 600 μm measuring macropores showing osteoinductive properties. Microsc. Res. Tech. 76:370–380, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructural investigation of intact orbital implant surfaces using atomic force microscopy

This study examined the surface nanostructures of three orbital implants: nonporous poly(methyl methacrylate) (PMMA), porous aluminum oxide and porous polyethylene. The morphological characteristics of the orbital implants surfaces were observed by atomic force microscopy (AFM). The AFM topography, phase shift and deflection images of the intact implant samples were obtained. The surface of the nonporous PMMA implant showed severe scratches and debris. The surface of the aluminum oxide implant showed a porous structure with varying densities and sizes. The PMMA implant showed nodule nanostructures, 215.56 ± 52.34 nm in size, and the aluminum oxide implant showed crystal structures, 730.22 ± 341.02 nm in size. The nonporous PMMA implant showed the lowest roughness compared with other implant biomaterials, followed by the porous aluminum oxide implant. The porous polyethylene implant showed the highest roughness and severe surface irregularities. Overall, the surface roughness of orbital implants might be associated with the rate of complications and cell adhesion. SCANNING 33: 211–221, 2011. © 2011 Wiley Periodicals, Inc.     

Reflectance confocal‐laser‐scanning microscopy in vivo assessments of cigarette‐induced dynamic alterations of cutaneous microcirculation on histomorphological level

Objective: Until now, high resolution reflectance confocal‐laser‐scanning microscopy (CLSM) was used for observation of cutaneous morphology in vivo and in real time. We hypothesized that CLSM also allows observation of dynamic processes of cutaneous microcirculation. Methods: Reflectance CLSM (Vivascope1500; Lucid, Rochester, NY) was performed in 24 young male habitual smokers (23 years, range: 19–26, body mass index 23.9 ± 4.04) with relatively limited cigarette exposure (mean: 3.1 ± 2.4 pack‐years). Eight matched nonsmokers served as controls. The quantitative blood cell flow and the diameter of capillary loops were determined prior (baseline), during, as well as 5 and 10 min after smoking. Results: Baseline value for blood cell flow was 55.50 ± 2.33 cells/min, and decreased over 45% during smoking (30.43 ± 3.76/min; P = 0.02). They were still 22% lower (43.33 ± 2.45/min; P = 0.01) 5 min after smoking and exceeded baseline values 10 min after smoking by 13% (63.00 ± 3.10/min; P > 0.05). The baseline values for capillary loop diameter (9.03 ± 0.22 μm) decreased by 21% (7.18 ± 0.28 μm; P = 0.03) during smoking, remained about 9% (8.23 ± 0.18 μm; P = 0.01) lower 5 min after smoking and exceeded baseline values insignificantly by 4% (9.38 ± 0.28 μm; P > 0.05) 10 min after smoking. There were no significant differences to the controls. Conclusion: Reflectance CLSM enables qualitative and quantitative observation of dynamic processes of cutaneous microcirculation on histomorphological level. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Effect of different implant abutment surfaces on OBA‐09 epithelial cell adhesion

For the long‐term success of implants, it is necessary to achieve a direct contact between the implant and the subjacent bone. To avoid bacterial penetration that could adversely affect the initial wound healing as well as the long‐term behavior of the implants, an early tissue barrier must form that is able to protect the biological peri‐implant structures. Given the need of an effective tissue early barrier around dental implants, the present study evaluated, in vitro, the influence of physical and chemical characteristics of two implant abutment surfaces on gingival epithelial cells (OBA‐9) adhesion. To this end, titanium (Ti) and zirconia (ZrO₂) disk‐shaped specimens were used mimicking the abutment components surfaces, while bovine enamel (BE) and glass cover slips (GCS) disks served as positive and negative controls, respectively. Roughness and surface free energy (SFE) of all materials were evaluated previously to cellular adhesion step. In sequence, the effect of each material on cells morphology and viability was analyzed after 1 and 24 hr. The results showed that roughness and SFE had no effect on the cell viability data or on their interaction (p = .559), independent of a post‐contact analysis of 1 or 24 hr. However, cells attachment and spreading increased after 24 hr on Ti and ZrO₂ than BE, corresponding to the highest SFE values. SFE appears to be an important property interfering on the quality of the soft tissue surrounding dental implants. These data can be considered a trigger point for developing new material surfaces.     

In vivo response to HA-polyhydroxybutyrate/polyhydroxyvalerate composite

This study examined the morphological and compositional structure of bone–implant interfaces after in vivo implantation into the tibias of rabbits. The implants were composed of biodegradable polyhydroxybutyrate/polyhydroxyvalerate copolymer reinforced with synthetic hydroxyapatite (HA) particles. Optical and scanning electron microscopy techniques were used, including energy‐dispersive X‐ray analysis. The interface was found to be morphologically, biologically and chemically active throughout the period of study. There was a strong tendency to rebuild the bone structure at the interface after implantation, independent of the composition of the implant, but direct bone bonding with the implant depended on the bioactive nature of the interface, as represented by the HA particles. At all implantation times, lamellar bone formed at the interface and replaced degrading polymer matrix, while engulfing HA filler particles. In regions about 50–100 from the interface, the bone region displayed an osteon organization. Osteoblasts and osteocytes were identified throughout the interface region. The thickness of the newly formed bone significantly increased over the period of the experiment from about 130 µm at 1 month to about 770 µm at 6 months. Materials that behave in this manner may be useful in some bone replacement therapies.     

Histological analysis of the alterations on cortical bone channels network after radiotherapy: A rabbit study

The aim of this study was to evaluate the effects of radiotherapy in cortical bone channels network. Fourteen rabbits were divided in two groups and test group received single dose of 15 Gy cobalt‐60 radiation in tibia, bilaterally. The animals were sacrificed and a segment of tibia was removed and histologically processed. Histological images were taken and had their bone channels segmented and called regions of interest (ROI). Images were analyzed through developed algorithms using the SCILAB mathematical environment, getting percentage of bone matrix, ROI areas, ROI perimeters, their standard deviations and Lacunarity. The osteocytes and empty lacunae were also counted. Data were evaluated using Kolmogorov‐Smirnov, Mann Whitney, and Student's t test (P < 0.05). Significant differences in bone matrix percentage, area and perimeters of the channels, their respective standard deviations and lacunarity were found between groups. In conclusion, the radiotherapy causes reduction of bone matrix and modifies the morphology of bone channels network. Microsc. Res. Tech. 73:1015–1018, 2010. © 2010 Wiley‐Liss, Inc.     

Morphological and structural study of pseudowollastonite implants in bone

In vitro experiments show that pseudowollastonite (α-CaSiO₃) is a highly bioactive material that forms a hydroxyapatite surface layer on exposure to simulated body fluid and also to human parotid saliva. This finding is very significant, as it indicates that the pseudowollastonite can be physically and chemically integrated into the structure of living bone tissue, and therefore could be suitable for repair or replacement of living bone.   The physical and chemical nature of the remodelled interface between the pseudowollastonite implants and the surrounding bone has been studied after in vivo implantation of 20 pseudowollastonite cylinders into rat tibias. The interfaces formed after 3, 6, 8 and 12 weeks of implantation were examined histologically using an optical microscope and also by analytical scanning electron microscopy.   SEM and X-ray elemental analysis showed that the new bone was growing in direct contact with the implants. Other examinations found that the bone was fully mineralized. The ionic exchange taking place at the implant interface with the body fluids was essential in the process of the implant integration through a dissolution–precipitation–transformation mechanism. The study found the interface biologically and chemically active over the 12-week implantation period. The rate of new bone formation decreased after the first 3 weeks and reached constant value over the following 9 weeks. The osteoblastic cells migrated towards the interface and colonized the surface at the contact areas with the cortical regions and also bone marrow.     

Micro‐Computed Tomography and Scanning Electron Microscopy Comparisons of Two Nickel–Titanium Rotary Root Canal Instruments Used With Reciprocating Motion

The single‐file root canal instrumentation technique using reciprocating motion has been gaining concern. Therefore, the purpose of this study was to compare the shaping ability of single ProTaper F2 file and WaveOne Primary file when they were used in the curved root canal with reciprocation motion and to investigate the durability of the file after use with a scanning electron microscopy (SEM). Changes in structure model index (SMI), root canal volume, curvature, surface area, and degree of transportation were measured from the cross‐sectional images of the prepared canals using the micro‐CT system with an isotropic resolution of 16 μm. Results showed that there were no differences in the changes of root canal volume, surface area, and SMI between the two file groups after the preparation (p > 0.05). The ProTaper group showed a curvature straightening value of 25.45 ± 12.51%, while the WaveOne group showed 27.30 ± 10.91%, and there was no statistically significant difference (p > 0.05). The transportation values between the two groups showed no significant differences (p > 0.05). SEM revealed that 60% of ProTaper files showed initiation of microcracks on the surface while those were detected on the only one WaveOne file. The single‐file technique using either WaveOne Primary or ProTaper F2 can be safely used under each reciprocating motion without creating an increased apical transportation in curved canals. However, the metallurgic property resists cyclic fatigue was more favorable with WaveOne under the scanning evaluation. SCANNING 35: 112‐118, 2013. © 2012 Wiley Periodicals, Inc.     

In situ assessment of the renal microcirculation in mechanically ventilated rats using sidestream dark‐field imaging

For microcirculation research there is a need for baseline data and feasibility protocols describing microcirculation of various organs. The aim of our study was to examine the reliability and reproducibility of sidestream dark‐field (SDF) imaging within the renal cortical microcirculation in rats. Renal microcirculation was observed using SDF probe placed on the exposed renal surface via the upper midline laparotomy. Video sequences recorded intermittently in short apneic pauses were analyzed off‐line by using AVA 3.0 software (MicroVision Medical, Amsterdam, the Netherlands). Results are expressed as mean (SD) or median (25–75% percentiles). We obtained 60 clear sequences from all recorded analyzable videos from all the animals. The total small vessel and all vessel density (in mm.mm^–2) were (28.79 ± 0.40) and (28.95 ± 0.40), respectively. The perfused small and all vessel density were (28.79 ± 0.40) and (28.95 ± 0.40), respectively. The DeBacker Score was (19.14 ± 0.43), the proportion of perfused vessels was 100% (100–100%) and the microvascular flow index was 3.49 (3–3.75). We conclude SDF imaging provides a reliable method to examine the renal microvascular bed in vivo and thus can be used for the study of the renal cortical vascular network in various experimental diseases models and clinical settings.     

Effect of different irrigation protocols on the radicular dentin interface and bond strength with a metacrylate‐based endodontic sealer

This study assessed the influence of different endodontic chemical substances on the adhesion of the Epiphany SE/Resilon system (with and without resinous solvent) to radicular dentin walls, using the push‐out test and scanning electron microscopy (SEM). Forty‐eight root canals of human canines were prepared biomechanically with ProTaper rotary files (crown‐down technique) and the radicular dentin was treated with either 17% EDTA, 2% chlorhexidine gel (CHX) or 2.5% NaOCl (control). The root canals were filled with Resilon cones and Epiphany SE sealer with and without resinous solvent. Six groups of eight canals each had their roots sectioned transversally to obtain 1‐mm thick slices. Data were subjected to statistical analysis by ANOVA and Tukey's tests. The specimens treated with 17% EDTA (1.59 ± 0.91) presented higher bond strength (P < 0.05) than those treated with 2.5% NaOCl (0.93 ± 0.27) and 2% CHX (0.92 ± 0.22). Significantly higher bond strength (P < 0.05) was observed when the Epiphany SE was prepared with (1.37 ± 0.78) than without (0.92 ± 0.33) solvent. Adhesive failures were predominant in all groups. SEM analysis showed greater homogeneity of the filling mass when the solvent was added to the sealer. Treatment of root canal walls with 17% EDTA, and addition of a resinous solvent to Epiphany SE produced the highest adhesion to radicular dentin. Microsc. Res. Tech. 77:446–452, 2014. © 2014 Wiley Periodicals, Inc.     

Short‐Term Response of Human Osteoblast‐Like Cells on Titanium Surfaces With Micro‐ and Nano‐Sized Features

Since the way that human bone cells behave on contact with different surfaces topographies seems to be crucial to osseointegration, the aim of the present study is to evaluate the participation of some micro‐ and nanosized features of Ti surfaces in the short‐term response of primary human osteoblast‐like cells (HOC). Surfaces were prepared as ground (G‐Ti), hydrofluoric acid etched (HF‐Ti), and sandblasted/HF‐etched (SLA‐Ti), and analyzed using both three‐dimensional (3D) profilometer and atomic force microscope (AFM). Cell morphology was assessed using scanning electron microscopy (SEM) after 4 and 24 h in culture. Cell viability, adhesion, and spreading were also evaluated 4 and 24 h after seeding over each surface. Data were compared by analysis of variance (ANOVA) complemented by Duncan test. Cell morphology, cell counting, and membrane integrity (Neutral Red, NR) were not affected by surface treatment at any time. However, HF‐Ti presented the smallest surface area and did not increase tetrazolium hydroxide (XTT) reduction from 4 to 24 h. On the other hand, a higher level of spreading was only found on the rougher and isotropic SLA‐Ti at 4 h. In conclusion, although all evaluated Ti surfaces allowed HOC short‐term adhesion, the finer topography introduced by HF as single treatment did not favor HOC mitochondrial activity and spreading. The rougher and more complex SLA surface seems to provide a better substrate for HOC short‐term response. SCANNING 34: 378‐386, 2012. © 2012 Wiley Periodicals, Inc.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.