Chiral Supramolecular Nanofibers Regulated Tumor-Derived Exosomes Secretion for Constructing an Anti-Tumor Extracellular Microenvironment.

Tumor-derived exosomes (TDEs) induced extracellular microenvironment has recently been validated to be critical for tumor progression and metastasis, however, remodeling it for oncotherapy still remains a major challenge due to difficulty in regulation of TDEs secretion. Herein, the supramolecular chiral nanofibers, composed of L/D-phenylalanine derivates (L/D-Phe) and linear hyaluronic acid (HA), are successfully employed to construct TDEs induced anti-tumor extracellular microenvironment. The left-handed L-Phe @HA nanofibers significantly inhibit TDEs secretion into extracellular microenvironment, which results in suppression of tumor proliferation and metastasis in vitro and vivo. Biological assays and theoretical modeling reveal that these results are mainly attributed to strong adsorption of the key exosomes transporters (Ras-related protein Rab-27A and synaptosome-associated protein 23) on left-handed L-Phe @HA nanofibers via enhanced stereoselective interaction, leading to degradation and phosphorylated dropping of exosomes transporters. Subsequently, transfer function of exosomes transporters is limited, which causes remarkable inhibition of TDEs secretion. These findings provide a promising novel insight of chiral functional materials to establish an anti-tumor extracellular microenvironment via regulation of TDEs secretion.

Assembly of Helical Nanostructures: Solvent-Induced Morphology Transition and Its Effect on Cell Adhesion.

Being able to precisely manipulate both the morphology and chiroptical signals of supramolecular assemblies will help to better understand the natural biological self-assembly mechanism. Two simple l/d-phenylalanine-based derivatives (L/DPFM) have been designed, and their solvent-dependent morphology evolutions are illustrated. It was found that, as the content of H2 O in aqueous ethanol solutions was increased, LPFM self-assembles first into right-handed nanofibers, then flat fibrous structures, and finally inversed left-handed nanofibers. Assemblies in ethanol and H2 O exhibit opposite conformations and circular dichroism (CD) signals even though they are constructed from the same molecules. Thus, the morphology-dependent cell adhesion and proliferation behaviors are further characterized. Left-handed nanofibers are found to be more favorable for cell adhesion than right-handed nanostructures. Quantitative AFM analysis showed that the L929 cell adhesion force on left-handed LPFM fibers is much higher than that on structures with inversed handedness. Moreover, the value of cell Young's modulus is lower for left-handed nanofibrous films, which indicates better flexibility. The difference in cell-substrate interactions might lead to different effects on cell behavior.

Realizing Abundant Chirality Inversion of Supramolecular Nanohelices by Multiply Manipulating the Binding Sites in Molecular Blocks.

The induction of diverse chirality regulation in nature by multiple binding sites of biomolecules is ubiquitous and plays an essential role in determining the biofunction of biosystems. However, mimicking this biological phenomenon and understanding at a molecular level its mechanism with the multiple binding sites by establishing an artificial system still remains a challenge. Herein, abundant chirality inversion is achieved by precisely and multiply manipulating the co-assembled binding sites of phenylalanine derivatives (D/LPPF) with different naphthalene derivatives (NA, NC, NP, NF). The amide and hydroxy group of naphthalene derivatives prefer to bind with the carboxy group of LPPF, while carboxylic groups and fluoride atoms tend to bind with the amide moiety of LPPF. All these diverse interaction modes can precisely trigger helicity inversion of LPPF nanofibers. In addition, synergistically manipulating the carboxy and amide binding sites from a single LPPF molecule to simultaneously interact with different naphthalene derivatives leads to chirality regulation. Typically, varying the solvent may switch the interaction modes and the switched new interaction modes can be employed to further regulate the chirality of the LPPF nanofibers. This study may provide a novel approach to explore chirality diversity in artificial systems by regulating the intermolecular binding sites.

Kinetic Co-assembly Pathway Induced Chirality Inversion Along with Morphology Transition.

Kinetic co-assembly pathway induced chirality inversion along with morphology transition is of importance to understand biological processes, but still remains a challenge to realize in artificial systems. Herein, helical nanofibers consisting of phenylalanine-based enantiomers (L/DPF) successfully transform into kinetically trapped architectures with opposite helicity through a kinetic co-assembly pathway. By contrast, the co-assemblies obtained by a thermodynamic pathway exhibit non-helical structures. The formation sequence of non-covalent interactions plays a crucial role in structural chirality of co-assemblies. For the kinetic pathway, the hydrogen bonding between D/LPF and naphthylamide derivatives forms before π-π stacking to facilitate the formation of helical structures with inverse handedness. This study may provide an approach to explore chirality inversion accompanied by morphology transition by manipulating the kinetic co-assembly pathway.

Supramolecular Hydrogels with Tunable Chirality for Promising Biomedical Applications.

Chirality exits from molecular-level, supramolecular, and nanoscaled helical structures to the macroscopic level in biological life. Among these various levels, as the central structural motifs in living systems (e.g., double helix in DNA, α-helix, ß-sheet in proteins), supramolecular helical systems arising from the asymmetrical spatial stacking of molecular units play a crucial role in a wide diversity of biochemical reactions (e.g., gene replication, molecular recognition, ion transport, enzyme catalysis, and so on). However, the importance of supramolecular chirality and its potential biofunctions has not yet been fully explored. Thus, generating chiral assembly to transfer nature's chiral code to artificial biomaterials is expected to be utilized for developing novel functional biomaterials. As one of the most commonly used biomaterials, supramolecular hydrogels have attracted considerable research interest due to their resemblance to the structure and function of the native extracellular matrix (ECM). Therefore, the performance and manipulation of chiral assembled nanoarchitectures in supramolecular hydrogels may provide useful insights into understanding the role of supramolecular chirality in biology.In this Account, recent progress on chiral supramolecular hydrogels is presented, including how to construct and regulate assembled chiral nanostructures in hydrogels with controllable handedness and then use them to develop chiral hydrogels that could be applied in biology, biochemistry, and medicine. First, a brief introduction is provided to present the basic concept related to supramolecular chirality and the importance of supramolecular chirality in living systems. The chiral assemblies in supramolecular hydrogels are strongly driven by noncovalent interactions between molecular building blocks (such as hydrogen bonding, π-π stacking, hydrophobic, and van der Waals interactions). Consequently, the handedness of these chiral assemblies can be regulated by many extra stimuli including solvents, temperature, pH, metal ions, enzymes, and photoirradiation, which is presented in the second section. This manipulation of the chirality of nanoarchitectures in supramolecular hydrogels can result in the development of potential biofunctions. For example, specific supramolecular chirality-induced biological phenomena (such as controlled cell adhesion, proliferation, differentiation, apoptosis, protein adsorption, drug delivery, and antibacterial adhesion) are presented in detail in the third section. Finally, the outlook of open challenges and future developments of this rapidly evolving field is provided. This account that highlights the diverse chirality-dependent biological phenomena not only helps us to understand the importance of chirality in life but also provides new ideas for designing and preparing chiral materials for more bioapplications.

Rational Fabrication of Multiple Dimensional Assemblies from Tryptophan-Based Racemate.

Fabricating structural complex assemblies from simple amino acid-based derivatives is attracting great research interests due to their easy accessibility and preparation. However, the morphological regulation of racemates (an equimolar mixture of enantiomers) were largely overlooked. In this work, through rational modulation of kinetic and thermodynamic parameters, we achieved multiple dimensional architectures employing tryptophan-based racemate (RPWM). Upon assembling, 1D bundled nanofibers, 2D lamellar nanostructure and 3D urchin-like microflowers could be obtained depending on the solvents used. The corresponding morphology evolutions were successfully illustrated by changing the enantiomeric excess (ee) value. Moreover, for RPWM, uniform 0D nanospheres were formed in H2 O under 4 °C, which could spontaneously convert into lamella under ambient temperature. Taking advantages of its temperature-responsive phase change behavior, RPWM assemblies exhibited excellent removal efficiency for organic dye RhB, and could be reused for several consecutive cycles without significant changes in its removal performance. Taken together, it's rational to envision that the engineering of racemates assembly pathways can greatly increase the robustness in a wide variety of supramolecular materials and further lead to their blooming versatile applications.

Effect of Stereochemistry on Chirality and Gelation Properties of Supramolecular Self-Assemblies.

Although chiral nanostructures have been fabricated at various structural levels, the transfer and amplification of chirality from molecules to supramolecular self-assemblies are still puzzling, especially for heterochiral molecules. Herein, four series of C2 -symmetrical dipeptide-based derivatives bearing various amino acid sequences and different chiralities are designed and synthesized. The transcription and amplification of molecular chirality to supramolecular assemblies are achieved. The results show that supramolecular chirality is only determined by the amino acid adjacent to the benzene core, irrespective of the absolute configuration of the C-terminal amino acid. In addition, molecular chirality also has a significant influence on the gelation behavior. For the diphenylalanine-based gelators, the homochiral gelators can be gelled through a conventional heating-cooling process, whereas heterochiral gelators form translucent stable gels under sonication. The racemic gels possess higher mechanical properties than those of the pure enantiomers. All of these results contribute to an increasing knowledge over control of the generation of specific chiral supramolecular structures and the development of new optimized strategies to achieve functional supramolecular organogels through heterochiral and racemic systems.

Solvent-Controlled Topological Evolution from Nanospheres to Superhelices.

Supramolecular assemblies with diverse morphologies are crucial in determining their biochemical or physical properties. However, the topological evolution and self-assembly intermediates as well as the mechanism remain elusive. Herein, a dynamic morphological evolution from solid nanospheres to superhelical nanofibers is revealed via self-assembly of a minimal l-tryptophan-based derivative (LPWM) with various mixed solvent combinations, including the formation of solid nanospheres, the fusion of nanospheres into pearling necklace, the disintegration of necklace into short nanofibers, the distortion of nanofibers into nanotwists, and the entanglement of nanotwists into superhelices. It is found that the breakage of intramolecular H-bonds and reconstruction of intermolecular H-bonds, as well as the variation of aromatic interactions and hydrophobic effects, are the key driving forces for topological transformation, especially the dimensional evolution. The nanospheres and nanofibers demonstrate discrepant behaviors towards mouse neural stem cell (NSC) differentiation that compared with negligible impact of nanospheres scaffold, the nanofibers scaffold is favorable for NSC differentiation into neurons. The remarkable dynamic regulation of assembly process, together with the NSC differentiation on twisted nanofibers are making this system an ideal model to interpret complex proteins fibrillation processes and investigate the structure-function relationship.

Visible Enantiomer Discrimination via Diphenylalanine-Based Chiral Supramolecular Self-Assembly on Multiple Platforms.

The development of enantioselective recognition is of great significance in medical science and pharmaceutical industry, which associates with the molecular recognition phenomenon widely observed in biological systems. In particular, the facile and straight achievement of visual enantioselective recognition has been drawing increasing consideration, but it is still a challenge. Herein, a heterochiral diphenylalanine-based gelator (LFDF) is synthesized, presenting left-handed nanofibers during self-assembly in ethanol, which accomplishes the phenylalaninol enantiomer recognition on multiple platforms. When adding l- or d-phenylalaninol into LFDF supramolecular solution followed by ultrasonic treatment, precipitate and gel are formed, respectively. Meanwhile, LFDF supramolecular gel completely collapses in a minute after dropping l-phenylalaninol, while the gel almost remains when d-type is employed. Moreover, a fluorescent supramolecular xerogel (ThT-LFDF) is fabricated by combining the LFDF gelator with thioflavine T (ThT), which could detect l-phenylalaninol accompanying with fluorescence quenching while d-type with barely decreasing. And the ThT-LFDF xerogel system shows a good sensitivity (reaches to ppm) for the detection of l-phenylalaninol. It is found that the chirality of the assembled nanofibers, as well as amino and carboxyl of phenylalaninol, plays a critical role on the discrimination process. The multiple and visible enantioselective recognition of phenylalaninol through chiral supramolecular self-assemblies shows potential applications in the fields of medical science and pharmaceutical industry.

The Cooperative Effect of Both Molecular and Supramolecular Chirality on Cell Adhesion.

Although helical nanofibrous structures have great influence on cell adhesion, the role played by chiral molecules in these structures on cells behavior has usually been ignored. The chirality of helical nanofibers is inverted by the odd-even effect of methylene units from homochiral l-phenylalanine derivative during assembly. An increase in cell adhesion on left-handed nanofibers and weak influence of cell behaviors on right-handed nanofibers are observed, even though both were derived from l-phenylalanine derivatives. Weak and negative influences on cell behavior was also observed for left- and right-handed nanofibers derived from d-phenylalanine, respectively. The effect on cell adhesion of single chiral molecules and helical nanofibers may be mutually offset.

In vivo and in vitro evaluation of effects of Mg-6Zn alloy on apoptosis of common bile duct epithelial cell.

Biodegradable magnesium alloy implants have attracted much attention because of their excellent biocompatibility and good mechanical properties. However, effects of Mg alloy on cell apoptosis remain unclear. The aim of this study was to investigate the effects of the Mg-6Zn alloy on the apoptosis and necrosis of common bile duct (CBD) epithelial cells. In the in vitro experiments, primary mouse extrahepatic bile epithelial cells (MEBECs) were exposed to Mg-6Zn alloy extracts with different concentrations (0, 40, 80, and 100 %). Flow cytometry analysis indicated that low concentration Mg-6Zn extract can induce apoptosis of MEBECs, and high concentration Mg-6Zn extracts may relate to necrosis and/or 'apoptotic necrosis'. Real-time PCR results showed that when MEBECs were treated with 40 % extracts for 3 days, the relative apoptotic genes including Bax, Bax/Bcl-2 ratio, NF-κB and caspase-3 were higher than those in the control group. In the in vivo experiments, Mg-6Zn alloy stents were implanted into rabbits' CBD for 1, 2, 3 weeks, respectively. Based on the hematoxylin and eosin (H&E) staining of peri-implant CBD tissue, no apoptotic bodies and necrotic cells were observed. Results of immunohistochemical staining also showed Mg-6Zn stents did not increase expression levels of apoptosis related gene such as Bax, Bcl-2, Bax/Bcl-2 ratio, TNF-α, NF-κB and caspase-3 in CBD, which indicating Mg-6Zn did not induce significant apoptosis in the in vivo experiments. The different results of in vitro and in vivo experiment may result from the low corrosion rate of Mg-6Zn alloy stents in vivo and local Mg(2+) ion concentration in CBD.

In vitro and in vivo assessment of the biocompatibility of an Mg-6Z(n) alloy in the bile.

There is a great clinical need for biodegradable bile duct stents. Biodegradable stents made of an Mg-6Zn alloy were investigated in both vivo animal experiment and in vitro cell experiments. During the in vivo experiments, blood biochemical tests were performed to determine serum magnesium, serum creatinine (CREA), blood urea nitro-gen (BUN), serum lipase (LPS), total bilirubin (TB) and glutamic-pyruvic transaminase (GPT) levels. Moreover, tissue samples of common bile duct (CBD), liver and kidney were taken for histological evaluation. In the in vitro experiments, primary mouse extrahepatic bile duct epithelial cells (MEBDECs) were isolated and cultured. Cytotoxicity testing was carried out using the MTT method. Flow cytometry analyses with propidium iodide staining were performed to evaluate the effect of Mg-6Zn alloy extracts on cell cycle. The in vivo experiments revealed no significant differences (P > 0.05) in serum magnesium, CREA, BUN, LPS, TB or GPT before and after the operation. Based on the HE results, hepatocytes, bile duct epithelial cells, renal glomerulus and renal tubule tissues did not present significant necrosis. In the in vitro experiments, the cell relative growth rate curve did not change significantly from 20 to 40 % extracts. In vitro experiments showed that 20-40 % Mg-6Zn extracts are bio-safe for MEBDECs. In vivo experiments showed that Mg-6Zn stents did not affect several important bio-chemical parameters or, harm the function or morphology of the CBD, kidney, pancreas and liver. Our data suggested that this Mg-6Zn alloy is a safe biocompatible material for CBD.

Shape and site dependent in vivo degradation of Mg-Zn pins in rabbit femoral condyle.

A type of specially designed pin model of Mg-Zn alloy was implanted into the full thickness of lesions of New Zealand rabbits' femoral condyles. The recovery progress, outer surface healing and in vivo degradation were characterized by various methods including radiographs, Micro-CT scan with surface rendering, SEM (scanning electron microscope) with EDX (Energy Dispersive X-ray analysis) and so on. The in vivo results suggested that a few but not sufficient bridges for holding force were formed between the bone and the implant if there was a preexisting gap between them. The rapid degradation of the implantation in the condyle would result in the appearance of cavities. Morphological evaluation of the specially designed pins indicated that the cusp was the most vulnerable part during degradation. Furthermore, different implantation sites with distinct components and biological functions can lead to different degradation rates of Mg-Zn alloy. The rate of Mg-Zn alloy decreases in the following order: implantation into soft tissue, less trabecular bone, more trabecular bone, and cortical bone. Because of the complexities of in vivo degradation, it is necessary for the design of biomedical Mg-Zn devices to take into consideration the implantation sites used in clinics.

Risk chain identification of single-vehicle accidents considering multi-risk factors coupling effect.

The real-time monitoring on the risk status of the vehicle and its driver can provide the assistance for the early detection and blocking control of single-vehicle accidents. However, complex risk coupling relationship is one of the main features of single-vehicle accidents with high mortality rate. On the basis of investigating the coupling effect among multi-risk factors and establishing a safety management database throughout the life cycle of vehicles, single-vehicle driving risk network (SVDRN) with a three-level threshold was developed, and its topology features were analyzed to assessment the importance of nodes. To avoid the one-sidedness of single indicator, the multi-attribute comprehensive evaluation model was applied to measure the comprehensive effect of characteristic indicators for nodes importance. A algorithm for real-time monitoring of vehicle driving risk status was proposed to identify key risk chains. The result revealed that improper operation, speeding, loss of vehicle control and inefficient driver management were the sequence of top four risk factors in the comprehensive evaluation result of nodes importance (mean value = 0.185, SD = 0.119). There were minor differences of 0.017 in the node importance among environmental factors, among which non-standard road alignment had the larger value. The improper operation and non-standard road alignment were the highest combination correlation of factors affecting road safety, with the support of 51.81% and the confidence of 69.35%. This identification algorithm of key risk chains that combines node importance and its risk state threshold can effectively determine the high-frequency risk transmission paths and risk factors through multi-vehicle test, providing a basis for centralization management of transport enterprises.

Chiral Supramolecular Hydrogel Enhanced Transdermal Delivery of Sodium Aescinate to Modulate M1 Macrophage Polarization Against Lymphedema.

Sodium aescinate (SA) shows great potential for treating lymphedema since it can regulate the expression of cytokines in M1 macrophages, however, it is commonly administered intravenously in clinical practice and often accompanied by severe toxic side effects and short metabolic cycles. Herein, SA-loaded chiral supramolecular hydrogels are prepared to prove the curative effects of SA on lymphedema and enhance its safety and transdermal transmission efficiency. In vitro studies demonstrate that SA- loaded chiral supramolecular hydrogels can modulate local immune responses by inhibiting M1 macrophage polarization. Typically, these chiral hydrogels can significantly increase the permeability of SA with good biocompatibility due to the high enantioselectivity between chiral gelators and stratum corneum and L-type hydrogels are found to have preferable drug penetration over D-type hydrogels. In vivo studies show that topical delivery of SA via chiral hydrogels results in dramatic therapeutic effects on lymphedema. Specifically, it can downregulate the level of inflammatory cytokines, reduce the development of fibrosis, and promote the regeneration of lymphatic vessels. This study initiates the use of SA for lymphedema treatment and for the creation of an effective chiral biological platform for improved topical administration.

Infected Diabetic Wound Regeneration Using Peptide-Modified Chiral Dressing to Target Revascularization.

Revascularization plays a critical role in the healing of diabetic wounds. Accumulation of advanced glycation end products (AGEs) and refractory multidrug resistant bacterial infection are the two major barriers to revascularization, directly leading to impaired healing of diabetic wounds. Here, an artfully designed chiral gel dressing is fabricated (named as HA-LM2-RMR), which consists of l-phenylalanine and cationic hexapeptide coassembled helical nanofibers cross-linked with hyaluronic acid via hydrogen bonding. This chiral gel possesses abundant chiral and cationic sites, not only effectively reducing AGEs via stereoselective interaction but also specifically killing multidrug resistant bacteria rather than host cells since cationic hexapeptides selectively interact with negatively charged microbial membrane. Surprisingly, the HA-LM2-RMR fibers present an attractive ability to activate sprouted angiogenesis of Human Umbilical Vein Endothelial Cells by upregulating VEGF and OPA1 expression. In comparison with clinical Prontosan Wound Gel, the HA-LM2-RMR gel presents superior healing efficiency in the infected diabetic wound with respect to angiogenesis and re-epithelialization, shortening the healing period from 21 days to 14 days. These findings for chiral wound dressing provide insights for the design and construction of diabetic wound dressings that target revascularization, which holds great potential to be utilized in tissue regenerative medicine.

Polydopamine-doped supramolecular chiral hydrogels for postoperative tumor recurrence inhibition and simultaneously enhanced wound repair.

Cancer recurrence remains a major challenge after primary tumor excision, and the inflammation of tumor-caused wounds can hinder wound healing and potentially promote tumor growth. Herein, a chiral L-phenylalanine-based (LPFEG) supramolecular hydrogel system encapsulated with polydopamine nanoparticles (PDA-NPs) has been developed in order to prevent tumor relapse after surgery and promote wound repair. PDA-NPs allow for near-infrared (NIR) light-triggered photothermal therapy, especially, it can scavenge free radicals in the surgical wound. LPFEG can mimic native extracellular matrix (ECM) structure to create a chiral microenvironment that enhances fibroblast adhesion, proliferation, and new tissue regeneration. With anticancer drug doxorubicin (DOX) loaded into the composite hydrogel, the antitumor effect is significantly enhanced by the integration of chemo-photothermal therapy both in vitro and in vivo. The PDA-based chiral supramolecular composite hydrogel as an effective postoperative adjuvant possesses promising applicable prospects in inhibiting tumor recurrence and accelerating wound healing after operation. STATEMENT OF SIGNIFICANCE: After primary tumor excision, cancer recurrence remains a severe concern, and the inflammation induced by tumor-related wounds can delay wound healing. Herein, we designed a chiral L-phenylalanine-based (LPFEG) supramolecular hydrogel platform that was co-assembled with polydopamine nanoparticles (PDA-NPs). Among them, PDA-NPs can offer photothermal therapy and scavenge free radicals in surgical wounds. LPFEG can create a chiral microenvironment that promotes fibroblast adhesion, proliferation, and new tissue regeneration. Furthermore, with anticancer drug doxorubicin (DOX) loaded into the composite hydrogel, the antitumor effect is considerably boosted. Therefore, the PDA-based chiral supramolecular hydrogel shows high application potential as a postoperative adjuvant in preventing tumor relapse as well as accelerating wound healing after surgery.

Chiral Supramolecular Hydrogel Loaded with Dimethyloxalyglycine to Accelerate Chronic Diabetic Wound Healing by Promoting Cell Proliferation and Angiogenesis.

Chronic refractory wounds are one of the most serious complications of diabetes, and the effects of common treatments are limited. Chiral hydrogel combined with dimethyloxalyglycine (DMOG) as a dressing is a promising strategy for the treatment of chronic wounds. In this research, we have developed a DMOG-loaded supramolecular chiral amino-acid-derivative hydrogel for wound dressings for full-thickness skin regeneration of chronic wounds. The properties of the materials, the ability of sustained release drugs, and the ability to promote angiogenesis were tested in vitro, and the regeneration rate and repair ability of full-thickness skin were tested in vivo. The chiral hydrogel had the ability to release drugs slowly. It can effectively promote cell migration and angiogenesis in vitro, and promote full-thickness skin regeneration and angiogenesis in vivo. This work offers a new approach for repairing chronic wounds completely through a supramolecular chiral hydrogel loaded with DMOG.

The bioactivated interfacial behavior of the fluoridated hydroxyapatite-coated mg-zn alloy in cell culture environments.

A partially fluorine substituted hydroxyapatite- (FHA-) coated Mg-Zn alloy was prepared to investigate the interfacial behavior of degradable Mg-based biomaterials with degradable bioactive coatings in a cell culture environment. Peaks from the results of X-ray diffraction (XRD) were characterized and compared before and after cell culture. It was found that Ca-P, including poorly crystalline ion-substituted Ca-deficient HA (CDHA), was formed in greater amounts on the interface of coated samples compared with the uncoated ones. A thermodynamic mechanism for Ca-P formation on biodegradable Mg alloys in a cell culture environment is proposed. Combined with improved cell calcification, the-FHA coated Mg alloys have the ability to promote CDHA formation, as expected thermodynamically. It is suggested that the specific cell culture environment and the bone-like FHA coatings together facilitate the observed behavior. Moreover, cell culture environment probably increased the biomineralization to a detectable level by affecting the kinetics of apatite formation.