Evaluation of Biological Response of STRO-1/c-Kit Enriched Human Dental Pulp Stem Cells to Titanium Surfaces Treated with Two Different Cleaning Systems.

Peri-implantitis-an infection caused by bacterial deposition of biofilm-is a common complication in dentistry which may lead to implant loss. Several decontamination procedures have been investigated to identify the optimal approach being capable to remove the bacterial biofilm without modifying the implant surface properties. Our study evaluated whether two different systems-Ni-Ti Brushes (Brush) and Air-Polishing with 40 µm bicarbonate powder (Bic40)-might alter the physical/chemical features of two different titanium surfaces-machined (MCH) and Ca++ nanostructured (NCA)-and whether these decontamination systems may affect the biological properties of human STRO-1+/c-Kit+ dental pulp stem cells (hDPSCs) as well as the bacterial ability to produce biofilm. Cell morphology, proliferation and stemness markers were analysed in hDPSCs grown on both surfaces, before and after the decontamination treatments. Our findings highlighted that Bic40 treatment either maintained the surface characteristics of both implants and allowed hDPSCs to proliferate and preserve their stemness properties. Moreover, Bic40 treatment proved effective in removing bacterial biofilm from both titanium surfaces and consistently limited the biofilm re-growth. In conclusion, our data suggest that Bic40 treatment may operatively clean smooth and rough surfaces without altering their properties and, consequently, offer favourable conditions for reparative cells to hold their biological properties.

Development of a novel method for amniotic fluid stem cell storage.

BACKGROUND AIMS: Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. METHODS: We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. RESULTS: hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. CONCLUSIONS: This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.

Human biliary tree stem/progenitor cells immunomodulation: Role of hepatocyte growth factor.

AIM: Human biliary tree stem/progenitor cells (hBTSC) are multipotent epithelial stem cells with the potential for allogenic transplant in liver, biliary tree, and pancreatic diseases. Human mesenchymal stem cells, but also epithelial stem cells, are able to modulate immune responses with different types of secretion molecules. METHODS: The initial aim of the present study was to develop for the first time a culture protocol in order to expand hBTSC in vitro through passages, allowing to maintain a similar stem cell and secretome profile. Furthermore, we investigated the secretome profile of the hBTSC to assess the production of molecules capable of affecting immune feedback. RESULTS: We found that hepatocyte growth factor produced by hBTSC exerts its cytoprotective role inducing apoptosis in human immune cells, such as lymphocytes. CONCLUSIONS: The present study, therefore, supports the hypothesis that hBTSC can be useful for the purpose of regenerative medicine, as they can be banked and expanded, and they can secrete immunoregulatory factors.

Apoptosis and inflammatory response in human astrocytes are induced by a transmissible cytotoxic agent of neurological origin.

We demonstrated the presence of an in vitro transmissible cytotoxic agent (TCA) in the cerebrospinal fluid (CSF) of patients with different acute neurological diseases. The nature of this agent is still a matter of study since repeated attempts have failed to identify it as a conventional infectious agent. Here, we describe the mechanisms through which TCA affects human astrocytes, demonstrating: a late apoptotic process, mediated by caspases 9 and 3 activation, involving the Bcl2-Bak-axis; an early and late p38 MAPK activation; an interference with the IL-8 and MCP-1 secretory response. These in vitro data provide initial evidence of TCA involvement as a pro-apoptotic and pro-inflammatory signal, directly affecting astrocytic behavior. The implications of these findings in certain neurological diseases will be discussed.

Different origin of adipogenic stem cells influences the response to antiretroviral drugs.

Lipodystrophy (LD) is a main side effect of antiretroviral therapy for HIV infection, and can be provoked by nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). LD exists in different forms, characterized by fat loss, accumulation, or both, but its pathogenesis is still unclear. In particular, few data exist concerning the effects of antiretroviral drugs on adipocyte differentiation. Adipose tissue can arise either from mesenchymal stem cells (MSCs), that include bone marrow-derived MSCs (hBM-MSCs), or from ectodermal stem cells, that include dental pulp stem cells (hDPSCs). To analyze whether the embryonal origin of adipocytes might impact the occurrence of different phenotypes in LD, we quantified the effects of several antiretroviral drugs on the adipogenic differentiation of hBM-MSCs and hDPSCs. hBM-MSCs and hDPSCs were isolated from healthy donors. Cells were treated with 10 and 50 µM stavudine (d4T), efavirenz (EFV), atazanavir (ATV), ritonavir (RTV), and ATV-boosted RTV. Viability and adipogenesis were evaluated by staining with propidium iodide, oil red, and adipoRed; mRNA levels of genes involved in adipocyte differentiation, i.e. CCAAT/enhancer-binding protein alpha (CEBPα) and peroxisome proliferator-activated receptor gamma (PPARγ), and in adipocyte functions, i.e. fatty acid synthase (FASN), fatty acid binding protein-4 (FABP4), perilipin-1 (PLIN1) and 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2), were quantified by real time PCR. We found that ATV, RTV, EFV, and ATV-boosted RTV, but not d4T, caused massive cell death in both cell types. EFV and d4T affected the accumulation of lipid droplets and induced changes in mRNA levels of genes involved in adipocyte functions in hBM-MSCs, while RTV and ATV had little effects. All drugs stimulated the accumulation of lipid droplets in hDPSCs. Thus, the adipogenic differentiation of human stem cells can be influenced by antiretroviral drugs, and depends, at least in part, on their embryonal origin.

Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations.

BACKGROUND: Human dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency. RESULTS: The STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs showed a slower proliferation, gradual loss of stemness, early cell senescence and apoptosis, compared to STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs. Both the subpopulations demonstrated similar abilities to differentiate towards mesoderm lineages, whereas a significant difference was observed after the neurogenic induction, with a greater commitment of STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs. Moreover, undifferentiated STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs did not show any expression of CD271 and nestin, typical neural markers, while STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs expressed both. CONCLUSIONS: These results suggest that STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs and STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs might represent two distinct stem cell populations, with different properties. These results trigger further analyses to deeply investigate the hypothesis that more than a single stem cell population resides within the dental pulp, to better define the flexibility of application of hDPSCs in regenerative medicine.

The Fas/Fas ligand apoptosis pathway underlies immunomodulatory properties of human biliary tree stem/progenitor cells.

BACKGROUND & AIMS: Human biliary tree stem/progenitor cells (hBTSCs) are multipotent epithelial stem cells, easily obtained from the biliary tree, with the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. Recent reports indicate that human mesenchymal stem cells are able to modulate the T cell immune response. However, no information exists on the capabilities of hBTSCs to control the allogeneic response. The aims of this study were to evaluate FasL expression in hBTSCs, to study the in vitro interaction between hBTSCs and human lymphocytes, and the role of Fas/FasL modulation in inducing T cell apoptosis in hBTSCs/T cell co-cultures. METHODS: Fas and FasL expression were evaluated in situ and in vitro by immunofluorescence and western blotting. Co-cultures of hBTSCs with human leukocytes were used to analyze the influence of hBTSCs on lymphocytes activation and apoptosis. RESULTS: hBTSCs expressed HLA antigens and FasL in situ and in vitro. Western blot data demonstrated that hBTSCs constitutively expressed high levels of FasL that increased after co-culture with T cells. Confocal microscopy demonstrated that FasL expression was restricted to EpCAM(+)/LGR5(+) cells. FACS analysis of T cells co-cultured with hBTSCs indicated that hBTSCs were able to induce apoptosis in activated CD4(+) and CD8(+) T cell populations. Moreover, the Fas receptor appears to be more expressed in T cells co-cultured with hBTSCs than in resting T cells. CONCLUSIONS: Our data suggest that hBTSCs could modulate the T cell response through the production of FasL, which influences the lymphocyte Fas/FasL pathway by inducing "premature" apoptosis in CD4(+) and CD8(+) T cells.

Human amniotic fluid stem cells: neural differentiation in vitro and in vivo.

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, ß-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression.

The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth, and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to identify those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and the lamin A precursor prelamin A are type V intermediate filament proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work, we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions and are therefore bona fide substrates of Akt. We report here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt also regulates Lmna transcription. Our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamin physiology to therapeutic approaches for lamin A-linked disorders.

A novel biomarker harvesting nanotechnology identifies Bak as a candidate melanoma biomarker in serum.

BACKGROUND: Melanoma represents only 4% of all skin cancers, but nearly 80% of skin cancer deaths. This manuscript applies several new measurement technologies with the purpose of elucidating molecular signatures of melanoma aggressiveness. PURPOSE: We sought to determine whether low-abundant serum proteins related to apoptotic pathways could be measured and correlated with defined melanoma subtypes. Hydrogel core shell nanoparticles, a new technology capable of selectively entrapping low molecular weight proteins and protecting them from enzymatic degradation, were used to capture candidate serum biomarkers. Biomarker levels were correlated with confocal microscopy, thereby representing a combination of new technologies for in vivo histologic documentation. RESULTS: Among a panel of analyzed serum proteins, Bak was differentially expressed between nevi and melanomas. Melanomas with higher Bak serum levels exhibited more pronounced junctional activity on confocal imaging, whereas lesions with 'sparse' dermal nests had weak Bak expression. CONCLUSIONS: Our study links serum proteome analysis with confocal microscopic clinical in vivo histologic classification of melanomas. Bak has not been previously measured in serum. Bak differential expression among melanoma subtypes confirms the importance of the apoptotic pathway as a contributor to melanoma aggressiveness.

Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs).

Human dental pulp stem cells (hDPSCs) are characterized by high proliferation rate, the multi-differentiation ability and, notably, low immunogenicity and immunomodulatory properties exerted through different mechanisms including Fas/FasL pathway. Despite their multipotency, hDPSCs require particular conditions to achieve chondrogenic differentiation. This might be due to the perivascular localization and the expression of angiogenic marker under standard culture conditions. FasL stimulation was able to promote the early induction of chondrogenic commitment and to lead the differentiation at later times. Interestingly, the expression of angiogenic marker was reduced by FasL stimulation without activating the extrinsic apoptotic pathway in standard culture conditions. In conclusion, these findings highlight the peculiar embryological origin of hDPSCs and provide further insights on their biological properties. Therefore, Fas/FasL pathway not only is involved in determining the immunomodulatory properties, but also is implicated in supporting the chondrogenic commitment of hDPSCs.

A-type lamins and signaling: the PI 3-kinase/Akt pathway moves forward.

Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A-linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A-Akt interaction and on the biological significance of their relationship.

Human MATER localization in specific cell domains of oocytes and follicular cells.

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.

Titanium Surface Properties Influence the Biological Activity and FasL Expression of Craniofacial Stromal Cells.

Mesenchymal stromal cells (MSCs) can be easily isolated form craniofacial bones during routine dentistry procedures. Due to their embryological origin from neural crest, they represent a suitable cell population to study cell-biomaterial interaction in the craniofacial field, including osteoinductive/osteointegrative processes. The biological and immunomodulatory properties of MSCs may be influenced by chemistry and topography of implant surfaces. We investigated if and how three different titanium surfaces, machined (MCH), sandblasted with resorbable blasting medium (RBM), and Ca++-nanostructured (NCA), may affect biological activity, osseointegration, and immunomodulatory properties of craniofacial MSCs. Cell proliferation, morphology, osteogenic markers, and FasL were evaluated on MSCs isolated from the mandibular bone after seeding on these three different surfaces. No statistically significant differences in cell proliferation were observed whereas different morphologies and growth patterns were detected for each type of surface. No difference in the expression of osteogenic markers was revealed. Interestingly, FasL expression, involved in the immunomodulatory activity of stem cells, was influenced by surface properties. Particularly, immunofluorescence analysis indicated that FasL expression increased on MCH surface compared to the others confirming the suggested role of FasL in promoting osteogenic differentiation. Titanium surface treatments and topography might reflect different biological behaviours of craniofacial MSCs and influence their osseointegration/immunomodulation properties.

Regenerative potential of human dental pulp stem cells in the treatment of stress urinary incontinence: In vitro and in vivo study.

OBJECTIVES: To evaluate the regenerative potential of human dental pulp stem cells (hDPSCs) in an animal model of stress urinary incontinence (SUI). SUI, an involuntary leakage of urine, is due to physical stress involving an increase in bladder pressure and a damage of external urethral sphincter affecting muscles and nerves. Conventional therapies can only relieve the symptoms. Human DPSCs are characterized by peculiar stemness and immunomodulatory properties and might provide an alternative tool for SUI therapy. MATERIALS AND METHODS: In vitro phase: hDPSCs were induced towards the myogenic commitment following a 24 hours pre-conditioning with 5-aza-2'-deoxycytidine (5-Aza), then differentiation was evaluated. In vivo phase: pudendal nerve was transected in female rats to induce stress urinary incontinence; then, pre-differentiated hDPSCs were injected in the striated urethral sphincter. Four weeks later, urethral sphincter regeneration was assayed through histological, functional and immunohistochemical analyses. RESULTS: Human DPSCs were able to commit towards myogenic lineage in vitro and, four weeks after cell injection, hDPSCs engrafted in the external urethral sphincter whose thickness was almost recovered, committed towards myogenic lineage in vivo, promoted vascularization and an appreciable recovery of the continence. Moreover, hDPSCs were detected within the nerve, suggesting their participation in repair of transected nerve. CONCLUSIONS: These promising data and further investigations on immunomodulatory abilities of hDPSCs would allow to make them a potential tool for alternative therapies of SUI.

Propagation of a transmissible cytotoxic activity on cultures of human peripheral blood lymphocytes.

Positive results were attained when human peripheral blood lymphocytes (PBLs) were investigated for their ability to propagate a transmissible cytotoxic activity (TCA) isolated on VERO cell cultures from a sample of cerebrospinal fluid (CSF) drawn from a woman with ischemic brain injury. In consideration of this finding it can be assumed that "in vivo" blood lymphocytes contributed to give rise to the TCA detected "in vitro" in the CSF inoculum.

Anterior Capsule of the Lens: Comparison of Morphological Properties and Apoptosis Induction following FLACS and Standard Phacoemulsification Surgery.

PURPOSE: Comparative evaluation of morphological features of anterior capsules and apoptosis induction in epithelial cells after femtosecond laser-assisted cataract surgery (FLACS) and standard phacoemulsification surgery. METHODS: Group 1: 30 FLACS anterior capsulotomies and Group 2: 30 manual anterior continuous curvilinear capsulorhexes. All patients were operated on by the same experienced surgeon. Morphological features of the anterior capsules and apoptosis induction in epithelial cells were evaluated. RESULTS: All patients revealed a significant mean best-corrected visual acuity (BCVA) improvement 3 months after surgery, and no major intraoperative nor postoperative complications occurred. The capsular epithelium appeared to be preserved in both groups. Scanning electron microscopy analysis revealed irregular saw-tooth shaped edges in capsules from Group 1 whereas capsules from Group 2 showed regular and smooth edges. A statistically significant higher expression of the downstream apoptotic effector cleaved caspase 3 was observed in Group 1. CONCLUSIONS: The saw-tooth appearance was likely due to the progressive sequence of laser pulses on the capsule. The low energy/high frequency properties of the laser pulse, combined with an overlapped pulse pattern, resulted in highly continuous morphology of capsule edges. The higher apoptosis induction in FLACS group might be due to photodisruption-dependent plasma generation and formation of cavitation bubbles.

Use of a 3D Floating Sphere Culture System to Maintain the Neural Crest-Related Properties of Human Dental Pulp Stem Cells.

Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties.

Human dental pulp stem cells expressing STRO-1, c-kit and CD34 markers in peripheral nerve regeneration.

Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The application of stem cells able to differentiate in Schwann cell-like cells in vitro and in vivo, could represent an attractive therapeutic approach for the treatment of nerve injuries. Further, stem cells sources sharing the same embryological origin as Schwann cells might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal subpopulation of human STRO-1+ /c-Kit+ /CD34+ DPSCs, expressing P75NTR , nestin and SOX-10, to differentiate into Schwann cell-like cells in vitro and to promote axonal regeneration in vivo, which led to functional recovery as measured by sustained gait improvement, in animal rat model of peripheral nerve injury. Transplanted human dental pulp stem cells (hDPSCs) engrafted into sciatic nerve defect, as revealed by the positive staining against human nuclei, showed the expression of typical Schwann cells markers, S100b and, noteworthy, a significant number of myelinated axons was detected. Moreover, hDPSCs promoted axonal regeneration from proximal to distal stumps 1 month after transplantation. This study demonstrates that STRO-1+ /c-Kit+ /CD34+ hDPSCs, associated with neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues. Copyright © 2016 John Wiley & Sons, Ltd.

Osteogenic Differentiation of hDPSCs on Biogenic Bone Apatite Thin Films.

A previous study reported the structural characterization of biogenic apatite (BAp) thin films realized by a pulsed electron deposition system by ablation of deproteinized bovine bone. Thin films annealed at 400°C exhibited composition and crystallinity degree very close to those of biogenic apatite; this affinity is crucial for obtaining faster osseointegration compared to conventional, thick hydroxyapatite (HA) coatings, for both orthopedics and dentistry. Here, we investigated the adhesion, proliferation, and osteogenic differentiation of human dental pulp stem cells (hDPCS) on as-deposited and heat-treated BAp and stoichiometric HA. First, we showed that heat-treated BAp films can significantly promote hDPSC adhesion and proliferation. Moreover, hDPSCs, while initially maintaining the typical fibroblast-like morphology and stemness surface markers, later started expressing osteogenic markers such as Runx-2 and OSX. Noteworthy, when cultured in an osteogenic medium on annealed BAp films, hDPSCs were also able to reach a more mature and terminal commitment, with respect to HA and as-deposited films. Our findings suggest that annealed BAp films not only preserve the typical biological properties of stemness of, hDPSCs but also improve their ability of osteogenic commitment.