Osteoblastic SaOS-2 Cells Research Articles

In vitro cytotoxicity and corrosion studies of some copper base shape memory alloys

In recent times Cu-based shape memory alloys are being used more extensively in many engineering applications due to their ease of production and processing and lower cost. But Cu-based alloys have not been found suitable for medical applications due to the toxic effects arising from the release of Cu ions into the body. The present work explores the feasibility of using Cu-based shape memory alloys (Cu-Al-Mn and Cu-Al-Ni) for biomedical applications. The toxicity level of the alloys was measured by cell culture (24 h) studies using SaOS-2 osteoblast and L6 myoblast cells, and other studies by using L6 myoblast cells (48 h and 72 h). Moreover, surface energy evaluation and corrosion studies were carried out in a simulated uterine fluid. An E-SEM was used to study the adhesion capability of the cells. The surface energy studies were carried out to assess the effect of cell adhesion on the alloy surface, while the corrosion studies to validate the cell culture results. Attempts were made to analyze the cause of toxic effects by analysing the polarization curves, results of SEM/EDAX and spectroscopy (ICP-OES) after corrosion studies in a simulated uterine fluid. It was found that Cu-Al-Mn alloys exhibit much better cell compatibility as compared to Cu-Al-Ni alloys.

Zanthoxylum piperitum reversed alveolar bone loss of periodontitis via regulation of bone remodeling-related factors

Ethnopharmacological relevanceZanthoxylum piperitum (ZP) has been used to prevent toothache in East Asia. Aim of studyIn this study, we investigated the effects of ZP on periodontitis along with alveolar bone loss. Materials and methodsTwenty-eight male Sprague-Dawley rats were assigned into 4 groups; non-ligated (NOR), ligated and treated vehicle (CTR), ligated and treated 1mg/mL ZP (ZP1), and ligated and treated 100mg/mL ZP (ZP100). Sterilized 3-0 nylon ligature was placed into the subgingival sulcus around the both sides of mandibular first molar. After topical application of 1 and 100mg/mL ZP for 2 weeks, mandibles was removed for histology. In addition, SaOS-2 osteoblast cells were treated 1, 10 and 100μg/mL ZP for 24h to analyze the expressions of alveolar bone-related markers. ResultsSeveral alveolar bone resorption pits, which indicate cementum demineralization were decreased by ZP treatment. Topical ZP treatment inhibited periodontitis-induced alveolar bone loss. In addition, there were significant reduction of osteoclastic activities following topical ZP treatment in periodontium. The expression of RANKL was decreased in SaOS-2 osteoblast cells by treating ZP, while that of OPG was increased. ZP treatment increased the expressions of Runx2 and Osterix in SaOS-2 cells. ConclusionIn summary, ZP treatment inhibited alveolar bone loss as well as maintained the integrity of periodontal structures via regulation of bone remodeling. ZP may be a therapeutic target for treating periodontitis.

Nanodiamonds on tetrahedral amorphous carbon significantly enhance dopamine detection and cell viability

We hypothesize that by using integrated carbon nanostructures on tetrahedral amorphous carbon (ta-C), it is possible to take the performance and characteristics of these bioelectrodes to a completely new level. The integrated carbon electrodes were realized by combining nanodiamonds (NDs) with ta-C thin films coated on Ti-coated Si-substrates. NDs were functionalized with mixture of carboxyl and amine groups NDandante or amine NDamine, carboxyl NDvox or hydroxyl groups NDH and drop-casted or spray-coated onto substrate. By utilizing these novel structures we show that (i) the detection limit for dopamine can be improved by two orders of magnitude [from 10µM to 50nM] in comparison to ta-C thin film electrodes and (ii) the coating method significantly affects electrochemical properties of NDs and (iii) the ND coatings selectively promote cell viability. NDandante and NDH showed most promising electrochemical properties. The viability of human mesenchymal stem cells and osteoblastic SaOS-2 cells was increased on all ND surfaces, whereas the viability of mouse neural stem cells and rat neuroblastic cells was improved on NDandante and NDH and reduced on NDamine and NDvox. The viability of C6 cells remained unchanged, indicating that these surfaces will not cause excess gliosis. In summary, we demonstrated here that by using functionalized NDs on ta-C thin films we can significantly improve sensitivity towards dopamine as well as selectively promote cell viability. Thus, these novel carbon nanostructures provide an interesting concept for development of various in vivo targeted sensor solutions.

Biocompatibility of plasma-treated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanofiber mats modified by silk fibroin for bone tissue regeneration

The objective of this study was to produce biocompatible plasma-treated and silk-fibroin (SF) modified poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofiber mats. The mats were plasma-treated using O2 or N2 gas to increase their hydrophilicity followed by SF immobilization for the improvement of biocompatibility. Contact angle measurements and SEM showed increased hydrophilicity and no disturbed morphology, respectively. Cell proliferation assay revealed that SF modification together with N2 plasma (PS/N2) promoted higher osteoblastic (SaOs-2) cell viability. Although, O2 plasma triggered more mineral formation on the mats, it showed poor cell viability. Consequently, the PS/N2 nanofiber mats would be a potential candidate for bone tissue engineering applications.

C-Fos induction by gut hormones and extracellular ATP in osteoblastic-like cell lines.

It is widely accepted that the c-Fos gene has a role in proliferation and differentiation of bone cells. ATP-induced c-Fos activation is relevant to bone homeostasis, because nucleotides that are present in the environment of bone cells can contribute to autocrine/paracrine signalling. Gut hormones have previously been shown to have an effect on bone metabolism. In this study, we used the osteoblastic Saos-2 cell line transfected with a c-Fos-driven reporter stimulated with five gut hormones: glucose inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), ghrelin and obestatin, in the presence or absence of ATP. In addition, TE-85 cells were used to determine the time course of c-Fos transcript induction following stimulation with GLP-1, and GLP-2 with or without ATP, using reverse transcription qPCR. The significant results from the experiments are as follows: higher level of c-Fos induction in presence of GIP, obestatin (p=0.019 and p=0.011 respectively), and GIP combined with ATP (p<0.001) using the luciferase assay; GLP-1 and GLP-2 combined with ATP (p=0.034 and p=0.002, respectively) and GLP-2 alone (p<0.001) using qPCR. In conclusion, three of the gut peptides induced c-Fos, providing a potential mechanism underlying the actions of these hormones in bone which can be directed or enhanced by the presence of ATP.

Bioactivity and Mechanical Stability of 45S5 Bioactive Glass Scaffolds Based on Natural Marine Sponges.

Bioactive glass (BG) based scaffolds (45S5 BG composition) were developed by the replica technique using natural marine sponges as sacrificial templates. The resulting scaffolds were characterized by superior mechanical properties (compression strength up to 4MPa) compared to conventional BG scaffolds prepared using polyurethane (PU) packaging foam as a template. This result was ascribed to a reduction of the total scaffold porosity without affecting the pore interconnectivity (>99%). It was demonstrated that the reduction of total porosity did not affect the bioactivity of the BG-based scaffolds, tested by immersion of scaffolds in simulated body fluid (SBF). After 1day of immersion in SBF, a homogeneous CaP deposit on the surface of the scaffolds was formed, which evolved over time into carbonate hydroxyapatite(HCA). Moreover, the enhanced mechanical properties of these scaffolds were constant over time in SBF; after an initial reduction of the maximum compressive strength upon 7days of immersion in SBF (to 1.2±0.2MPa), the strength values remained almost constant and higher than those of BG-based scaffolds prepared using PU foam (<0.05MPa). Preliminary cell culture tests with Saos-2 osteoblast cell line, namely direct and indirect tests, demonstrated that no toxic residues remained from the natural marine sponge templates and that cells were able to proliferate on the scaffold surfaces.

Development and characterization of a xenograft material from New Zealand sourced bovine cancellous bone.

A xenograft (bovine hydroxyapatite [BHA]) was developed from New Zealand sourced bovine cancellous bone by a successful defatting and deproteinizing procedure. The BHA was chemically, compositionally and structurally characterized. Fourier transform infrared spectroscopy confirmed the removal of organic matter from the bone matrix and the presence of carbonate ( CO32-), hydroxyl (OH- ), and phosphate ( PO43-) functional groups. X-ray diffraction analysis suggested that the processed bone corresponds characteristically to hydroxyapatite (HA). SEM analysis showed that the BHA has an interconnected porous architecture with a pore diameter ranging from 100 to 700 μm while µCT analysis calculated the total porosity as 73.46% ± 1.08. Furthermore, the BHA was stable up to 1000°C and lost only 1.8% of its weight. The Ca/P molar ratio of the BHA was 1.58, which is comparable with commercially available natural HA-Endobon® . After 28 days of incubation in simulated body fluid (SBF), the pH value only fluctuated between 7.1 and 7.5 and the BHA scaffold did not degrade significantly by weight indicating the scaffold had excellent chemical and structural stability. In vitro studies showed the BHA was cytocompatible and supported the proliferative growth of Saos-2 osteoblast cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1054-1062, 2017.

Manipulation of Cell Proliferation and Migration Employing Surface Acoustic Waves and Hydrophobic/Hydrophilic Structured Substrates

Tissue engineering for the purpose of nerve or spinal cord repair has become an emerging topic in biophysical and medical research. Addressing this issue relies on an understanding of the principles of tissue growth and applying this understanding to produce functional replacement tissues for clinical use after injury. During wound healing, the damaged tissue layer is reassembled and regenerated through cell proliferation and migration. Thus, any type of external stimuli being able to improve this process of cell regeneration could be highly useful for accelerating wound repair. To study such an external wound healing parameter, we use surface acoustic waves (SAW) for dynamic in vitro manipulation of living cells in a precise controlled manner. In our investigations, we use osteoblastic SaOs-2 cells on a Si02-covered piezoelectric LiNbO3 substrate which we irradiate with SAW continuously for 72hours. Employing a conventional wound healing assay, we show the SAW - treated cells exhibit significantly increased proliferation and migration, respectively, as compared to control samples. Apart from quantifying our experimental findings, we also demonstrate the biocompatibility and biofunctionality of our SAW reactor by using LDH assays. We can neglect parasitic or beneficial side effects such as changing substrate temperature or nutrient flow rate through (OR ‘by closely’) monitoring the temperature and flow field using infrared microscopy and micro particle image velocimetry. Indeed, our results show that the SAW-induced dynamic, mechanical, and electrical stimulation directly promotes cell growth by increasing cell regeneration and migration rates. Our next goal is to guide the cells along certain tracks using hydrophobic/hydrophilic trails in combination with SAW.

The endocrine disruptor cadmium alters human osteoblast-like Saos-2 cells homeostasis in vitro by alteration of Wnt/β-catenin pathway and activation of caspases

The pollutant Cadmium (Cd) is widespread in the environment and causes alterations of human health by acting as an endocrine disruptor. Bone tissue seems to be a crucial target of Cd contamination. Indeed, we have previously demonstrated that this endocrine disruptor induces osteoblast apoptosis and necrosis. Thus, aim of this study was to further evaluate the effect of Cd on osteoblasts homeostasis, investigating potential modification of the Wnt/β-catenin intracellular pathway, the intracellular process involved in programmed cellular death and the cytoskeletal alterations. To this purpose, human osteoblastic Saos-2 cells, a human osteosarcoma osteoblast-like cell line, were cultured and treated with Cd. Osteoblastic cells were treated for 6 h with 10μM Cd, which induced nuclear translocation of β-catenin and increased expression of Wnt/β-catenin target genes. Longer exposure to the same Cd concentration induced osteoblastic cell apoptosis. To better characterize the intracellular events involved in these Cd-induced alterations, we evaluated the effect of Cd exposure on actin filaments and proteins associated to cytoskeletal actin, characterized by the presence of LIM domains. Long (15, 24 h) exposure of osteoblasts to Cd reduced LIM proteins expression and induced actin filaments destruction and a significant caspase-3 activation after 24 h. In addition, to prove that Cd induces osteoblastic cells apoptosis after long exposure, we performed TUNEL assay which demonstrated increase of cell apoptosis after 24 h. In conclusion, our study shows that osteoblasts exposed to Cd for short intervals of time demonstrated an increase in cell proliferation through a Wnt/β-catenin dependent mechanism, likely as a compensatory mechanism in response to cell injury. Longer exposure to the same Cd concentration induced cells apoptosis through cytoskeleton disruption-mediated mechanisms and caspase activation.

Cell adhesion on Ti surface with controlled roughness

In this report, the in situ interaction between Saos-2 osteoblast cells and a smooth Ti surface was examined over time. The adhesion kinetics and mechanisms of cellular proliferation were monitored by quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). The rate of Saos-2 attachment on Ti surfaces, obtained from the measurements performed with the QCM, is a first-order reaction, with k=2.10−3 min−1. The impedance measurements indicate that in the absence of cells, the Ti resistance diminishes over time (7 days), due to the presence of amino acids and proteins from the culture medium that have been a dsorbed, while in the presence of osteoblasts, this decrease is much greater because of the compounds generated by the cells that accelerate the dissolution of Ti.

Osteoblastic cells trigger gate currents on nanocrystalline diamond transistor

We show the influence of osteoblastic SAOS-2 cells on the transfer characteristics of nanocrystalline diamond solution-gated field-effect transistors (SGFET) prepared on glass substrates. Channels of these fully transparent SGFETs are realized by hydrogen termination of undoped diamond film. After cell cultivation, the transistors exhibit about 100× increased leakage currents (up to 10nA). During and after the cell delamination, the transistors return to original gate currents. We propose a mechanism where this triggering effect is attributed to ions released from adhered cells, which depends on the cell adhesion morphology, and could be used for cell culture monitoring.

In Vitro Biocompatibility and Antibacterial Activity of Electrospun Ag Doped HAp/PHBV Composite Nanofibers

The authors report herein in vitro antibacterial property and osteoblast biocompatibility of electrospun Ag doped HAp/PHBV (Ag-HAp/PHBV) composite nanofibers as an osteoconductive and antibacterial material for bone tissue engineering applications. Ag-HAp powders were synthesized and stable composite suspensions of Ag-HAp/PHBV were prepared with the aid of a cationic surfactant DTAB for the electrospinning process. Continuous and uniform composite nanofibers were generated within a diameter range of 400–900 nm. Obtained nanocomposite scaffolds provide a favorable environment for bone mineralization, SaOS-2 osteoblastic cell attachment and growth as well as they present antibacterial activity against E. coli and S. aureus bacteria without any noticeable cytotoxic effect.

Radiopacity and cytotoxicity of Portland cement associated with niobium oxide micro and nanoparticles.

Objective Mineral Trioxide Aggregate (MTA) is composed of Portland Cement (PC) and bismuth oxide (BO). Replacing BO for niobium oxide (NbO) microparticles (Nbµ) or nanoparticles (Nbη) may improve radiopacity and bioactivity. The aim of this study was to evaluate the radiopacity and cytotoxicity of the materials: 1) PC; 2) White MTA; 3) PC+30% Nbµ; 4) PC+30% Nbη.Material and Methods For the radiopacity test, specimens of the different materials were radiographed along an aluminum step-wedge. For cell culture assays, Saos-2 osteoblastic-cells (ATCC HTB-85) were used. Cell viability was evaluated through MTT assay, and bioactivity was assessed by alkaline phosphatase activity assay.Results The results demonstrated higher radiopacity for MTA, followed by Nbµ and Nbη, which had similar values. Cell culture analysis showed that PC and PC+NbO associations promoted greater cell viability than MTA.Conclusions It was concluded that the combination of PC+NbO is a potential alternative for composition of MTA.

Cytocompatibility, gene-expression profiling, apoptotic, mechanical and (29)Si, (31)P solid-state nuclear magnetic resonance studies following treatment with a bioglass-chitosan composite.

The performance therapy of chitosan (CH)-doped bioactive glass (BG) has been evaluated in vitro and in vivo. In vitro, the effect of CH-BG was assessed on human Saos-2 osteoblast cells. In vivo, Wistar rats were ovariectomized (OVX) and CH, BG and CH-BG were implanted in bone tissue. After 3 days of CH-BG contact, cell viability of Saos-2 osteoblast increased by 16.4% as compared to the control group. The runt-related transcription factor 2 (RUNX2/Cbfa1) and osteocalcin (OC) gene expressions were significantly increased with 600 and 300%, respectively, in contact of CH-BG as compared with CH. In vivo, the apoptotic index in the OVX-CH-BG group was decreased by 80%. A mechanical hardness test showed a significant bone strength improvement after CH-BG implantation (40%). The CH-BG composite may therefore prove clinically useful as a bioactive bone substitute.

Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone

Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were assessed. Finally, the cultivation of osteoblastic SaOS-2 cells on the surface-functionalized CHS-g-PCL was followed by analyses of cell viability, mineral deposition and alkaline phosphatase activity. These characterizations revealed a composite that combines the versatile properties of CHS-g-PCL with the osteogenic activity of the silicatein/biosilica coating and, hence, represents an innovative alternative to conventionally used CHS/PCL composites for biomedical applications, where stable bone–material interfaces are required.

Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70) family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER)-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip), which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of potential clinical significance for understanding the cellular and molecular bases of amelogenin-induced periodontal tissue regeneration.

Suspension thermal spraying of hydroxyapatite: Microstructure and in vitro behaviour

In cementless fixation of metallic prostheses, bony ingrowth onto the implant surface is often promoted by osteoconductive plasma-sprayed hydroxyapatite coatings. The present work explores the use of the innovative High Velocity Suspension Flame Spraying (HVSFS) process to coat Ti substrates with thin homogeneous hydroxyapatite coatings.The HVSFS hydroxyapatite coatings studied were dense, 27–37μm thick, with some transverse microcracks. Lamellae were sintered together and nearly unidentifiable, unlike conventional plasma-sprayed hydroxyapatite. Crystallinities of 10%–70% were obtained, depending on the deposition parameters and the use of a TiO2 bond coat. The average hardness of layers with low (<24%) and high (70%) crystallinity was ≈3.5GPa and ≈4.5GPa respectively. The distributions of hardness values, all characterised by Weibull modulus in the 5–7 range, were narrower than that of conventional plasma-sprayed hydroxyapatite, with a Weibull modulus of ≈3.3.During soaking in simulated body fluid, glassy coatings were progressively resorbed and replaced by a new, precipitated hydroxyapatite layer, whereas coatings with 70% crystallinity were stable up to 14days of immersion. The interpretation of the precipitation behaviour was also assisted by surface charge assessments, performed through Z-potential measurements.During in vitro tests, HA coatings showed no cytotoxicity towards the SAOS-2 osteoblast cell line, and surface cell proliferation was comparable with proliferation on reference polystyrene culture plates.

Challenges in the Characterization of Plasma-Processed Three-Dimensional Polymeric Scaffolds for Biomedical Applications

Low-temperature plasmas offer a versatile method for delivering tailored functionality to a range of materials. Despite the vast array of choices offered by plasma processing techniques, there remain a significant number of hurdles that must be overcome to allow this methodology to realize its full potential in the area of biocompatible materials. Challenges include issues associated with analytical characterization, material structure, plasma processing, and uniform composition following treatment. Specific examples and solutions are presented utilizing results from analyses of three-dimensional (3D) poly(ε-caprolactone) scaffolds treated with different plasma surface modification strategies that illustrate these challenges well. Notably, many of these strategies result in 3D scaffolds that are extremely hydrophilic and that enhance human Saos-2 osteoblast cell growth and proliferation, which are promising results for applications including tissue engineering and advanced biomedical devices.

Preparation, characterization and biological test of 3D-scaffolds based on chitosan, fibroin and hydroxyapatite for bone tissue engineering

This work describes the preparation and characterization of porous 3D-scaffolds based on chitosan (CHI), chitosan/silk fibroin (CHI/SF) and chitosan/silk fibroin/hydroxyapatite (CHI/SF/HA) by freeze drying. The biomaterials were characterized by X-ray diffraction, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy and energy dispersive spectroscopy. In addition, studies of porosity, pore size, contact angle and biological response of SaOs-2osteoblastic cells were performed. The CHI scaffolds have a porosity of 94.2±0.9%, which is statistically higher than the one presented by CHI/SF/HA scaffolds, 89.7±2.6%. Although all scaffolds were able to promote adhesion, growth and maintenance of osteogenic differentiation of SaOs-2 cells, the new 3D-scaffold based on CHI/SF/HA showed a significantly higher cell growth at 7days and 21days and the level of alkaline phosphatase at 14 and 21days was statistically superior compared to other tested materials.

Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66GPa)≫dispersed (9.99kPa)>central region (5.20kPa)>peripheral regions (3.67kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed>central region>peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25–40kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site.