Osteogenic Differentiation Conditions Research Articles

Osteogenic potential of apical papilla stem cells mediated by platelet-rich fibrin and low-level laser

To evaluate the osteogenic potential of platelet-rich fibrin (PRF) and low-level laser therapy (LLLT) on human stem cells from the apical papilla (SCAP) we isolated, characterized, and then cultured in an osteogenic medium cells with PRF and/or LLLT (660 nm, 6 J/m2-irradiation). Osteogenic differentiation was assessed by bone nodule formation and expression of bone morphogenetic proteins (BMP-2 and BMP-4), whereas the molecular mechanisms were achieved by qRT-PCR and RNA-seq analysis. Statistical analysis was performed by ANOVA and Tukey’s post hoc tests (p < 0.05* and p < 0.01**). Although PRF and LLLT increased bone nodule formation after 7 days and peaked at 21 days, the combination of PRF + LLLT led to the uppermost nodule formation. This was supported by increased levels of BMP-2 and -4 osteogenic proteins (p < 0.005). Furthermore, the PRF + LLLT relative expression of specific genes involved in osteogenesis, such as osteocalcin, was 2.4- (p = 0.03) and 28.3- (p = 0.001) fold higher compared to the PRF and LLLT groups, and osteopontin was 22.9- and 1.23-fold higher, respectively (p < 0.05), after 7 days of interaction. The transcriptomic profile revealed that the combination of PRF + LLLT induces MSX1, TGFB1, and SMAD1 expression, after 21 days of osteogenic differentiation conditions exposition. More studies are required to understand the complete cellular and molecular mechanisms of PRF plus LLLT on stem cells. Overall, we demonstrated for the first time that the combination of PRF and LLLT would be an excellent therapeutic tool that can be employed for dental, oral, and craniofacial repair and other tissue engineering applications.

Preparation, characterization and biocompatibility of calcium peroxide-loaded polycaprolactone microparticles.

To explore the physicochemical characteristics and biocompatibility of calcium peroxide (CPO)-loaded polycaprolactone (PCL) microparticle. The CPO/PCL particles were prepared. The morphology and elemental distribution of CPO, PCL and CPO/PCL particles were observed with scanning electron microscopy and energy dispersive spectroscopy, respectively. Rat adipose mesenchymal stem cells were isolated and treated with different concentrations (0.10%, 0.25%, 0.50%, 1.00%) of CPO or CPO/PCL particles. The mesenchymal stem cells were cultured in normal media or osteogenic differentiation media under the hypoxia/normoxia conditions, and the amount of released O2 and H2O2 after CPO/PCL treatment were detected. The gene expressions of alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), osteopontin (OPN) and osteocalcin (OCN) were detected by realtime RT-PCR. SD rats were subcutaneously injected with 1.00% CPO/PCL particles and the pathological changes and infiltration of immune cells were observed with HE staining and immunohistochemistry at day 7 and day 14 after injection. Scanning electron microscope showed that CPO particles had a polygonal structure, PCL particles were in a small spherical plastic particle state, and CPO/PCL particles had a block-like crystal structure. Energy dispersive spectroscopy revealed that PCL particles showed no calcium mapping, while CPO/PCL particles showed obvious and uniform calcium mapping. The concentrations of O2 and H2O2 released by CPO/PCL particles were lower than those of CPO group, and the oxygen release time was longer. The expressions of Alp, Runx2, Ocn and Opn increased with the higher content of CPO/PCL particles under hypoxia in osteogenic differentiation culture and normal culture, and the induction was more obvious under osteogenic differentiation conditions (all P<0.05). HE staining results showed that the muscle tissue fibers around the injection site were scattered and disorderly distributed, with varying sizes and thicknesses at day 7 after particle injection. Significant vascular congestion, widened gaps, mild interstitial congestion, local edema, inflammatory cell infiltration, and large area vacuolization were observed in some tissues of rats. At day 14 after microparticle injection, the muscle tissue around the injection site and the tissue fibers at the microparticle implantation site were arranged neatly, and the gap size was not thickened, the vascular congestion, local inflammatory cell infiltration, and vacuolization were significantly improved compared with those at day 7. The immunohistochemical staining results showed that the expressions of CD3 and CD68 positive cells significantly increased in the surrounding muscle tissue, and were densely distributed in a large area at day 7 after particle injection. At day 14 of microparticle injection, the numbers of CD3 and CD68 positive cells in peripheral muscle tissue and tissue at the site of particle implantation were lower than those at day 7 (all P<0.01). CPO/PCL particles have good oxygen release activity, low damage to tissue, and excellent biocompatibility.

Characterization and Study of Gene Expression Profiles of Human Periodontal Mesenchymal Stem Cells in Spheroid Cultures by Transcriptome Analysis.

A spheroid is known as a three-dimensional culture model, which better simulates the physiological conditions of stem cells. This study is aimed at identifying genes specifically expressed in spheroid-cultured human periodontal ligament mesenchymal stem cells (hPDLMSCs) using RNA-seq analysis to evaluate their functions. Transcriptome analysis was performed using spheroid and monolayer cultures of hPDLMSCs from four patients. Cluster and Gene Ontology analyses revealed that genes involved in cell-cell adhesion as well as the G2/M and G1/S transitions of mitotic cell cycles were strongly expressed in the monolayer culture group. However, genes involved in the negative regulation of cell proliferation, histone deacetylation, and bone morphogenetic protein signaling were strongly expressed in the spheroid culture group. We focused on the transcription factor nuclear receptor subfamily 4 group A member 2 (NR4A2) among the genes that were strongly expressed in the spheroid culture group and analyzed its function. To confirm the results of the transcriptome analysis, we performed real-time polymerase chain reaction and western blotting analyses. Interestingly, we found that the mRNA and protein expressions of NR4A2 were strongly expressed in the spheroid-cultured hPDLMSCs. Under osteogenic differentiation conditions, we used siRNA to knock down NR4A2 in spheroid-cultured hPDLMSCs to verify its role in osteogenesis. We found that NR4A2 knockdown significantly increased the levels of mRNA expression for osteogenesis-related genes alkaline phosphatase (ALP), Osteopontin (OPN), and type 1 collagen (COL1) (Student's paired t-test, p < 0.05). ALP activity was also significantly increased when compared to the negative control group (Student's paired t-test, p < 0.05). Additionally, spheroid-cultured hPDLMSCs transfected with siNR4A2 were cultured for 12 days, resulting in the formation of significantly larger calcified nodules compared to the negative control group (Student's paired t-test, p < 0.05). On the other hand, NR4A2 knockdown in hPDLMSC spheroid did not affect the levels of chondrogenesis and adipogenesis-related genes under chondrogenic and adipogenic conditions. These results suggest that NR4A2 negatively regulates osteogenesis in the spheroid culture of hPDLMSCs.

Influence of gellan gum-hydroxyapatite spongy-like hydrogels on human osteoblasts under long-term osteogenic differentiation conditions

The scientific community has been doing significant efforts towards engineering new 3D bone models in recent years. Osteocytes are mechanosensitive cells that play significant roles in the maintenance of bone homeostasis. Currently, as far as we know, there are no 3D models that faithfully recapitulate a bone microenvironment capable of promoting the differentiation of osteoblasts towards osteocytes. Besides, in the existing models, the use of human cells does not prevail over the animal cell lines. For so, we propose a 3D model that may have important implications for ongoing efforts towards a better understanding of bone physiology and disease. The main aim of the current work was the promotion of an effective differentiation of osteoblasts into osteocytes by mean of using a 3D model composed of primary human osteoblasts (hOBs) cultured on Gellan Gum-Hydroxyapatite (GG-HAp) matrix under a long-term osteogenic culture. The results revealed that GG-HAp matrix stimulated a fast cell migration/entrapment, attachment, spreading, and mineralization. Moreover, the transition process from osteoblasts to osteocytes was confirmed by the expression of the osteogenic-related (ALP, Runx2, COL I, OC, OPN and OSX) and osteocyte-related (hPDPN) marker throughout the culture time. Overall, the developed 3D model holds a great promise for the treatment of various bone diseases, namely on diagnostic applications and for bone regeneration purposes.

Abstract MP08: Telomerase Reverse Transcriptase Mediates Osteogenesis In Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is the leading heart valve disorder in the US. It is characterized by an active accumulation of calcium nodules on the aortic valve leaflets which lead to stiffening and remodeling of the valve leaflets causing valve dysfunction, cardiac failure and increased stroke risk. Inflammation and mechanical stresses contribute to CAVD pathogenesis. However, the mechanisms driving the fibrocalcific remodeling of the aortic valve are currently ill-defined. Multiple studies have revealed that the catalytic subunit of telomerase reverse transcriptase (TERT) can induce gene transcription and its overexpression primes mesenchymal stem cells to differentiate into osteoblasts, suggesting that TERT has a role in the activation of osteogenic transcriptional programs. We hypothesized that TERT contributes to early events leading to calcification of the valve leaflet. In human calcified valve tissue, we found that TERT protein is highly expressed in areas of calcification compared to control valve tissue, with no effect on telomere length. Alpha-SMA, a VIC activation marker, and RUNX2, a key transcription factor involved in the osteogenic differentiation of osteoblast, were also elevated in CAVD tissue. Under osteogenic differentiation conditions, human valve interstitial cells (VICs) upregulated TERT, RUNX2, and alpha-SMA protein levels and calcified, while CAVD VICs calcified de novo. Inflammatory stimuli intensified in vitro calcification, and induced TERT, RUNX2, and alpha-SMA protein expression. PLA and ChIP analysis showed that TERT interacts with interacted with Signal Transducer and Activator of Transcription 5A/B (STAT5) and together bind to RUNX2 promoter, respectively. shRNA-mediated TERT downregulation reduced expression of RUNX2 and alpha-SMA and genetic deletion of Tert in murine mesenchymal stem cells and vascular smooth muscle cells prevented calcification. These data provide evidence that TERT is required for calcification, regulates the transition of quiescent VICs into calcifying VICs, and that STAT5 functions as a TERT-interacting partner for DNA binding.

Microstructures and biological properties of 3D-printed titanium intervertebral spacer with the tri-calcium phosphate loaded demineralized bone matrix hydrogel

The aim of this study was to improve the osteogenic ability of 3D-printed porous titanium (Ti) intervertebral spacer with a Tri-calcium Phosphate (TCP) loaded Demineralized Bone Matrix Hydrogel (DBM). Microstructures and morphologies of Ti, DBM loaded Ti (Ti/DBM), and TCP and DBM loaded Ti (Ti/DBM/TCP) scaffolds were characterized by SEM and EDX. In-vitro studies showed that Ti/DBM/TCP scaffold was biocompatible and could promote osteogenesis by up-regulating the expression of bone-related genes. Furthermore, the results of in-vivo studies using rabbit-femur defect model revealed that the implanted Ti/DBM/TCP scaffold had superior bone regeneration. The TCP and DBM loading were an effective approach of inducing osteogenesis in 3D-printed Ti intervertebral spacer by providing favorable osteogenic differentiation conditions and promoting bone formation.

Study of optimal conditions for growth and osteogenic differentiation of dental pulp stem cells based on glucose and serum content.

Dental pulp stem cells (DPSCs) have shown promising characteristics in terms of their proliferation and osteogenic differentiation potential, which could be of greater benefit in regenerative dentistry. However, obstacles remain in the in vitro cultivation of DPSCs, which significantly affect their growth and differentiating ability. Therefore in this study, we demonstrated the growth and osteogenic differentiation of DPSCs in the presence of media containing different combinations of serum and glucose to get an optimized combination of both. DPSCs were cultured in media containing combinations of low glucose (LG), low serum (LS), high glucose (HG), and high serum (HS). The proliferation and osteogenic differentiation were assessed in DPSCs cultured with these different combinations of culture conditions. High glucose high serum condition significantly inhibited the proliferation of DPSCs and also affected their clonogenic potential, as evidenced by colony-forming units. Irrespective of the serum content, high glucose in the media also decreased the osteogenic potential of DPSCs confirmed by functional staining, and downregulation of osteogenesis-related genes. High glucose content in the culture media affects the growth and differentiation potential of the DPSCs. Hence, the culture conditions for the DPSCs should be reconsidered to utilize their maximum potential.

Abstract 15843: A Novel Role for Telomerase in Calcific Aortic Valve Disease

Introduction: Calcific aortic valve disease (CAVD) is a disorder characterized by the slow, but a progressive thickening of the aortic valve leaflet that develops into severe calcification. The only current therapy is aortic valve replacement. Telomerase is an enzymatic complex best known for its telomere-extending activities on the ends of chromosomes yet, the catalytic subunit (TERT) has been implicated in multiple non-canonical transcriptional and epigenetic activities, including priming of mesenchymal stem cells (MSCs) to differentiate into osteoblasts, and transcriptional regulation of inflammatory genes. Hypothesis: We hypothesize that non-canonical TERT activity contributes to the progression of CAVD. Methods: We performed biochemical assays to study the role of TERT in the calcification process using primary tissues and valve interstitial cells (VICs) from control and CAVD patients, smooth muscle cells (SMCs) from WT and Tert knockout mice, and mesenchymal stem cells (MSCs). Results: We found that TERT protein is highly expressed in calcified aortic valves and VICs isolated from patients with calcified valves, compared to healthy valves. VICs can be induced to calcify under osteogenic differentiation conditions, and we found that TERT accumulated after fourteen days in this culture, with no effect on either telomere length, proliferation, or senescence. We expanded the scope of our approach by evaluating TERT's influence on calcification of mice aortic smooth muscle cells (mSMCs). We found WT mSMCs readily calcified in vitro, but mSMCs from Tert knockout mice did not, and Tert deletion also reduced valve calcification in a Ldlr / Tert double knockout mice model compared to Ldlr knockout alone. In VICs, shRNA mediated TERT downregulation reduced expression of RUNX2 . Finally, we found that inflammatory signals intensify in vitro calcification, induce TERT expression, and we show evidence that TERT interacts with STAT5. Conclusion: Our data suggest that TERT is required for valve calcification by stimulating transcriptional pathways promoting the osteogenic transition of quiescent VICs into calcifying VICs in the aortic valve. These results indicate that TERT is an active contributor to the calcification process of valve tissues.

Acquisition of cancer stem cell properties in osteosarcoma cells by defined factors

BackgroundCancer stem cells (CSCs) are considered to be responsible for tumor initiation, formation, and poor prognosis of cancer patients. However, the rarity of CSCs in clinical samples makes it difficult to elucidate characteristics of CSCs, especially in osteosarcoma (OS). The aim of this study is to verify whether it is possible to generate CSC-like cells by transducing defined factors into an OS cell line.MethodsWe retrovirally transduced the Octamer-binding transcription factor 3/4 (OCT3/4), Kruppel-like factor 4 (KLF4), and SRY-box transcription factor 2 (SOX2) genes into the MG-63 human OS cell line (MG-OKS). Parental and GFP-transduced MG-63 cells were used as negative control. We assessed the properties of the generated cells in vitro and in vivo. Multiple comparisons among groups were made using a one-way analysis of variance (ANOVA) followed by post hoc testing with Tukey’s procedure.ResultsMG-OKS cells in vitro exhibited the significantly increased mRNA expression levels of CSC markers (CD24, CD26, and CD133), decreased cell growth, increased chemoresistance and cell migration, and enhanced sphere formation. Notably, MG-OKS cells cultured under osteogenic differentiation conditions showed strongly positive staining for both Alizarin Red S and alkaline phosphatase, indicating osteogenesis of the cells. Gene ontology analysis of microarray data revealed significant upregulation of epidermal-related genes. Tumors derived from MG-OKS cells in vivo were significantly larger than those from other cells in μCT analysis, and immunohistochemical staining showed that Ki-67, osteocalcin, and HIF-1α-positive cells were more frequently detected in the MG-OKS-derived tumors.ConclusionsIn this study, we successfully generated OS CSC-like cells with significantly enhanced CSC properties following transduction of defined factors.

Endocytic Trafficking of DMP1 and GRP78 Complex Facilitates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

Periodontal ligament contains periodontal ligament stem cells that maintain tissue homeostasis. Targeting hPDLSCs (human periodontal ligament cells) is a promising strategy for repair and regeneration of bone tissue destroyed by periodontal diseases. However, the mechanisms by which PDLSCs differentiate into osteoblasts to form a mineralized matrix is unclear. In this study, we demonstrate for the first time the molecular events that contribute to osteogenic differentiation of PDLSCs. Dentin matrix protein 1 (DMP1) and its receptor, Glucose regulated protein-78 (GRP78), are localized in the progenitor cells of the PDL. Our overall goal is to demonstrate the formation of DMP1-GRP78 complex at the plasma membrane and subsequent protein trafficking and nuclear localization to promote osteogenic differentiation. To study the internalization and routing of the complex, we mimic an in vivo differentiation scenario by stimulating cells with DMP1 and culturing them in the presence of osteogenic differentiation conditions. We first demonstrate the translocation of the ER chaperone protein GRP78 to the plasma membrane during the differentiation process. Total internal reflection microscopy imaging demonstrates the formation and internalization of the receptor- ligand (GRP78-DMP1) complex. Confocal microscopy results show the internalization of the GRP78-DMP1 complex specifically through the caveolin pathway and trafficked through the cell with various endocytic markers such as Rab5 and 7 GTPases to early and late endosomes respectively. DMP1 is ultimately transported to the nucleus where it functions to promote osteogenic differentiation as demonstrated by quantitative Real-Time PCR. This observation is the first report that suggests DMP1 and GRP78 can interact at the plasma membrane, then packaged in vesicles and ultimately DMP1 is routed to the nucleus where it aids in osteogenic differentiation of PDLSCs. Characterizing the osteogenic potential of PDLSCs would favor the development of therapeutic strategies for reconstruction of mineralized tissues destroyed by periodontal diseases.

Abstract 480: A Novel Role for Telomerase in Calcific Aortic Valve Disease

Intro: Telomerase (TERT) is an enzyme best known for its telomere-extending activities on the ends of chromosomes, however, less known are the non-canonical, transcriptional and epigenetic actitivies of TERT. We sought to assess if TERT, through non-canonical activities, contributes to the progression of calcific aortic valve disease (CAVD). It has been established that overexpression of TERT in mesenchymal stem cells primes these cells to differentiate into osteoblasts. Other studies have identified non-canonical roles for TERT in inducing the transcription of genes in inflammatory and cell differentiation pathways. Methods: Using mesenchymal stem cells, primary valve interstitial cells (VICs) from control and CAVD patients, and smooth muscle cells (SMCs) from WT and TERT knockout mice, we performed in vitro biochemical assays to study the role of TERT in the calcification process. Results: Our data shows that TERT is highly expressed in CAVD valves compared to Control valves. Under osteogenic differentiation conditions Control valve interstitial cells (VICs) upregulate TERT protein levels and calcify. Inflammatory signals induce TERT expression in VICs and exacerbates calcification. Similarly, WT vascular VSMCs readily calcify in vitro, but VSMCs from TERT knockout mice do not, and TERT deletion also reduced valve calcification in LDLR/TERT double knock mice. These studies provide evidence that TERT is necessary is the osteogenic switch of a healthy to a calcifying VIC. Knocking down TERT reduces expression of the osteogenic transcription factor RUNX2 and we further provided evidence that STAT5 may help to mediate TERTs effects on osteogenic gene transcription. Conclusion: From these data we suggest that TERT is required for valve calcification by inducing the osteogenic transition of quiescent valve interstitial cells (VICs) into calcifying VICs, and that TERT and STAT5 co-regulate transcription of osteogenic genes. These results indicate that TERT is an active contributor to the calcification process of valve tissues and our futures studies will delineate the mechanisms involved.

Oxidized alginate beads for tunable release of osteogenically potent mesenchymal stromal cells.

Bone defect repair can benefit from local delivery of mesenchymal stromal cells (MSCs). However, local harsh environmental conditions after injury may necessitate a cell therapy strategy that shields MSCs initially and releases them locally over time. This may be possible by using biomaterials that exhibit stimuli-responsive degradability, such as oxidized alginate hydrogels that undergo hydrolytic degradation. However, it remains unknown whether varying encapsulation periods compromise MSC osteogenic differentiation capacity after release. To address this, we cultured MSCs in 3D alginate beads with tunable degradability before characterizing the function of released cells. Alginates were oxidized to different degrees (2%, 3%, and 4%) to achieve distinct rates of degradation (days to weeks), then functionalized with RGD peptides to enable cell adhesion, and modified additionally with 6-aminofluorescin to enable fluorescence-based detection. Bead morphology, degradation kinetics, cell morphology, and cell release kinetics were monitored over time. Cells that were released from the beads were stimulated to differentiate into the osteogenic lineage. Our results indicate that MSCs released from all bead groups retained a strong ability to deposit mineralized matrix under osteogenic differentiation conditions. These findings provide the basis for designing and implementing biomaterial-based strategies for the in-situ temporal delivery of potent MSCs at bone defect sites.

Rational design of gelatin/nanohydroxyapatite cryogel scaffolds for bone regeneration by introducing chemical and physical cues to enhance osteogenesis of bone marrow mesenchymal stem cells.

Identification of key components in the chemical and physical milieu for directing osteogenesis is a requirement in the investigation of tissue engineering scaffolds for advancement of bone regeneration. In this study, we engineered different gelatin-based cryogels and studied the effect of nanohydroxyapatite (nHAP) and crosslinking agents on scaffold properties and its osteogenic response towards bone marrow stem cells (BMSCs). The cryogels examined are 5% gelatin and 5% gelatin/2.5% nHAP, crosslinked either with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) or glutaraldehyde (GA). We confirmed that nHAP or the crosslinking agent has no effects on scaffold pore size and porosity. Nonetheless, incorporation of nHAP increased mechanical strength, swelling ratio and degree of crosslinking, but decreased degradation rate. Cryogels crosslinked with EDC showed faster degradation and promoted osteogenic differentiation of BMSCs while those prepared from GA crosslinking promoted proliferation of BMSCs. Furthermore, osteogenic differentiation was always enhanced in the presence of nHAP irrespective of the culture medium (normal or osteogenic) used but osteogenic medium always provide a higher extent of osteogenic differentiation. Employing gelatin/nHAP cryogel crosslinked by EDC in a bioreactor for dynamic culture of BMSCs, cyclic compressive mechanical simulation was found to be beneficial for both cell proliferation and osteogenic differentiation. However, the optimum conditions for osteogenic differentiation and cell proliferation were found at 30% and 60% strain, respectively. We thus demonstrated that osteogenic differentiation of BMSCs could be tuned by taking advantages of chemical cues generated from scaffold chemistry or physical cues generated from dynamic cell culture in vitro. Furthermore, by combining the best cryogel preparation and in vitro cell culture condition for osteogenesis, we successfully employed in vitro cultured cryogel/BMSCs constructs for repair of rabbit critical-sized cranial bone defects.

Expression of Musashi-1 During Osteogenic Differentiation of Oral MSC: An In Vitro Study.

Background: Musashi-1 (MSI1) is a negative regulator of mesenchymal stromal cell (MSC) differentiation which in turn favors cell proliferation. However, little is known about its expression by MSC from the oral cavity and in the context of osteogenic differentiation. Aim: The aim of this study was to analyze the expression of MSI1 in the context of osteogenic differentiation of MSC derived from the oral cavity. Material/methods: For this in vitro study, MSC were isolated from six different origins of the oral cavity. They were extensively characterized in terms of proliferative and clonogenicity potential, expression of stemness genes (MYC, NANOG, POU5F1, and SOX2), expression of surface markers (CD73, CD90, CD105, CD14, CD31, CD34, and CD45) and adipo-, chondro- and osteogenic differentiation potential. Then, osteogenic differentiation was induced and the expression of MSI1 mRNA and other relevant markers of osteogenic differentiation, including RUNX2 and Periostin, were also evaluated. Results: Cell populations from the alveolar bone (pristine or previously grafted with xenograft), dental follicle, dental germ, dental pulp, and periodontal ligament were obtained. The analysis of proliferative and clonogenicity potential, expression of the stemness genes, expression of surface markers, and differentiation potential showed similar characteristics to those of previously published MSC from the umbilical cord. Under osteogenic differentiation conditions, all MSC populations formed calcium deposits and expressed higher SPARC. Over time, the expression of MSI1 followed different patterns for the different MSC populations. It was not significantly different than the expression of RUNX2. In contrast, the expression of MSI1 and POSTN and RUNX2 were statistically different in most MSC populations. Conclusion: In the current study, a similar expression pattern of MSI1 and RUNX2 during in vitro osteogenic differentiation was identified.

Does the Harvesting Site Influence the Osteogenic Potential of Mesenchymal Stem Cells?

Total hip arthroplasty (THA) represents one of the commonest surgical procedures in the orthopedic field. Osteointegration of the implant with native bone is essential for an optimal result; thus, the quality of the patient's bone surrounding the implant (i.e., the bone stock) is crucial. However, in some cases, the bone stock is insufficient and needs to be improved with autologous grafts rich in multipotent cells (i.e., from the iliac crest, from the head of the femur, or from the subchondral bone harvested from the acetabulum) or allogenic frozen bone. It is not known if the harvesting site may influence the osteogenic potential of these cells. Thus, our aim was to characterize and compare multipotent cells collected from the bone marrow, acetabular subchondral bone, and trabecular bone on the femoral head with a focus on osteogenic differentiation. The cells from three sources had a fibroblast-like phenotype and expressed surface antigens CD73, CD90, and CD105 and are negative to CD11b, CD34, and CD45. Although all these cells could be induced to differentiate into osteoblasts, chondrocytes, and adipocytes, they displayed different differentiation potentials. In osteogenic differentiation condition, the cells from the acetabulum had the lowest accumulation of calcium deposit while the cells originated from the bone marrow and femur created a considerably increased amount of the deposit. These findings were confirmed by quantitative polymerase chain reaction (qPCR). In chondrogenic and adipogenic conditions, bone marrow cells possessed a predominant differential capacity compared with the others, illustrated by high collagen type II expression together with a cartilage-like lacuna structure and the presence of fat-specific markers, respectively. To our knowledge, this is the first study comparing and demonstrating that the progenitor cells obtained from diverse surgical sites in hip replacement procedure share common characteristics of MSC but differ about plasticity and may provide rational for clinical application in cell therapy and bone grafting. The project number L1033 is registered with ClinicalTrials.gov NCT03369457.

Bone quality assessment of osteogenic cell cultures by Raman microscopy.

The use of autologous stem/progenitor cells represents a promising approach to the repair of craniofacial bone defects. The calvarium is recognized as a viable source of stem/progenitor cells that can be transplanted in vitro to form bone. However, it is unclear if bone formed in cell culture is similar in quality to that found in native bone. In this study, the quality of bone mineral formed in osteogenic cell cultures were compared against calvarial bone from postnatal mice. Given the spectroscopic resemblance that exists between cell and collagen spectra, the feasibility of extracting information on cell activity and bone matrix quality were also examined. Stem/progenitor cells isolated from fetal mouse calvaria were cultured onto fused-quartz slides under osteogenic differentiation conditions for 28 days. At specific time intervals, slides were removed and analyzed by Raman microscopy and mineral staining techniques. We show that bone formed in culture at Day 28 resembled calvarial bone from 1-day-old postnatal mice with comparable mineralization, mineral crystallinity, and collagen crosslinks ratios. In contrast, bone formed at Day 28 contained a lower degree of ordered collagen fibrils compared with 1-day-old postnatal bone. Taken together, bone formed in osteogenic cell culture exhibited progressive matrix maturation and mineralization but could not fully replicate the high degree of collagen fibril order found in native bone.

Fibrin-blood clot as an initial stage of formation of bone regeneration after a bone fracture

This study focuses on mechanisms which regulate the process of fracture healing. We studied the form and position of the fibrin-blood clots (FBC) in the zone near the fragments among patients with limb fractures: macroscopically (during open surgical operations to reposition bone fragments) and using sonography. We conducted a histological and immuno-histochemical analysis of biopsy material obtained from the zone around the fracture during surgical procedures on 16 patients in 1–18 days after the fracture. We determined that the density of FBC and their form depends on the volume of damage to the periosteum-muscular fascia and the extent of the shift of fragments. In most cases, with a closed fracture, fibrin-blood clots had a spindle-shaped form. Fibrin along the periphery and in the zone between the fragments has a dense structure, and becomes cellular in central zones. The cells surrounded by fibrin partitions contain blood cells and serum. In many places, fibrin partitions had a one-direction orientation position, and the cells were oval-elongated, which indicated hydrostatic pressure in them. Proliferation of mesenchymal cells began in the vital tissues around the FBC, then during reproduction they pentrated to fibrin. Lengthwise axis of the cells was parallel to fibrin partitions. The bone trabeculae which form on the third week after fracture repeated the orientation of the fibrin partitions. It was determined that the vascular endothelial growth factor (VGEF) concentrates in fibrin and remains in it over the first week after the fracture, later it was found in endotheliocytes, fibroblasts and osteoblasts. The process of filling of the cells with around-fracture FBC lasted 12–18 days and during this period, their osteogenic differentiation occurred. Such tempi and orientation of the process is caused by fibrin with a concentration of growth factors in it. Using the results of the study, it could be assumed that the main conditions for osteogenic differentiation of cells are high concentration of VGEF in the fibrin, which initiates neoangiogenesis and internal tension of fibrin partitions. The formation of structured FBC around the ends of the fragments, which contain VGEF should be considered an initial stage of the process of forming of bone regeneration.

Increased elastic modulus of plasma polymer coatings reinforced with detonation nanodiamond particles improves osteogenic differentiation of mesenchymal stem cells.

In the present study we demonstrated that composite PPHMDS/DND coatings with elastic moduli close to those of mature bone tissue (0.2-2.8 GPa) stimulated growth and osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs). Composite coatings were prepared by a method of plasma polymerization (PP) where detonation nanodiamond (DND) particles in different amounts (0.1, 0.5, and 1 mg/mL) were added to hexamethyldisiloxane (HMDS) before plasma deposition. This method allows variation only in the reduced elastic modulus (Er´) with increase in the particle concentration, while the other surface properties, including surface wettability and topography, did not change. The response of hAD-MSCs to the increasing stifness showed an effect on adhesion and osteogenic differentiation but not on cell proliferation. Matrix mineralization and cell spreading were maximized on PPHMDS/DND coatings with the highest elastic modulus (2.826 GPa), while the differences in proliferation rates among the samples were negligible. In general, PPHMDS/DND coatings provide better conditions for growth and osteogenic differentiation of hAD-MSCs in comparison to glass coverslips, confirming their suitability for osteo-integration applications. Additionally, our findings support the hypothesis that biomaterials with elasticity similar to that of the native tissue can improve the differentiation potential of mesenchymal stem cells.

Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells

Background: Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations. Purpose: To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation. Study Design: Controlled laboratory study. Methods: MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated. Results: The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics—(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)—and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 106 ± 3.7 × 105 and 1.6 × 106 ± 4.7 × 105 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin (P = .025), aggrecan (P < .0001), and collagen type 2 (P = .044) gene expression compared with hADSCs, respectively, after culture in differentiation medium. Moreover, both hAECs and hAMSCs produced significantly greater quantities of mineralized (P < .0001) and cartilaginous (P = .0004) matrix at earlier time points compared with hADSCs when cultured under identical osteogenic and chondrogenic differentiation conditions, respectively. Conclusion: Amnion-derived MSCs demonstrate a greater differentiation potential toward bone and cartilage compared with hADSCs. Clinical Relevance: Amniotic MSCs may be the source of choice in the regenerative treatment of bone or osteochondral musculoskeletal disease. They show significantly higher yields and better differentiation toward these tissues than MSCs derived from adipose.

ToF-SIMS study of differentiation of human bone-derived stromal cells: new insights into osteoporosis

Lipids have numerous important functions in the human body, as they form the cells' plasma membranes and play a key role in many disease states, presumably also in osteoporosis. Here, the fatty acid composition of the outer plasma membranes of cells differentiated into the osteogenic and adipogenic direction is studied with surface-sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). For data evaluation, principal component analysis (PCA) is applied. Human (bone-derived) mesenchymal stromal cells (hMSCs) from an osteoporotic donor and a control donor are compared to reveal differences in the fatty acid composition of the membranes. The chemical information is correlated to staining and real-time quantitative polymerase chain reaction (rt-qPCR) results to provide insight into the gene expression of several differentiation markers on the RNA level. Adipogenic differentiation of hMSCs from a non-osteoporotic donor correlates with increased relative intensities of all fatty acids under investigation. After osteogenic differentiation of non-osteoporotic cells, the relative mass signal intensities of unsaturated fatty acids such as oleic and linoleic acids are increased. However, the osteoporotic cells show increased levels of palmitic acid in the plasma membrane after exposure to osteogenic differentiation conditions, which correlates to an immature differentiation state relative to non-osteoporotic osteogenic cells. This immature differentiation state is confirmed by increased early osteogenic differentiation factor Runx2 on RNA level and by less calcium mineralization spots seen in von Kossa staining and ToF-SIMS images. Graphical abstract Time-of-flight secondary ion mass spectrometry is applied to analyze the fatty acid composition of the outer plasma membranes of cells differentiated into the adipogenic and osteogenic direction. Cells from an osteoporotic and a control donor are compared to reveal differences due to differentiation and disease stage of the cells.