Jaw Periosteal Cells Research Articles

Three-Dimensionally Cultured Jaw Periosteal Cells Attenuate Macrophage Activation of CD4+ T Cells and Inhibit Osteoclastogenesis.

The implementation of a successful therapeutic approach that includes tissue-engineered grafts requires detailed analyses of graft-immune cell interactions in order to predict possible immune reactions after implantation. The phenotypic plasticity of macrophages plays a central role in immune cell chemotaxis, inflammatory regulation and bone regeneration. The present study addresses effects emanating from JPC-seeded β-TCP constructs (3DJPCs) co-cultivated with THP-1 derived M1/M2 macrophages within a horizontal co-culture system. After five days of co-culture, macrophage phenotype and chemokine secretion were analyzed by flow cytometry, quantitative PCR and proteome arrays. The results showed that pro-inflammatory factors in M1 macrophages were inhibited by 3DJPCs, while anti-inflammatory factors were activated, possibly affected by the multiple chemokines secreted by 3D-cultured JPCs. In addition, osteoclast markers of polarized macrophages were inhibited by osteogenically induced 3DJPCs. Functional assays revealed a significantly lower percentage of proliferating CD4+ T cells in the groups treated with secretomes from M1/M2 macrophages previously co-cultured with 3DJPCs compared to controls without secretomes. Quantifications of pit area resorption assays showed evidence that supernatants from 3DJPCs co-cultured with M1/M2 macrophages were able to completely suppress osteoclast maturation, compared to the control group without secretomes. These findings demonstrate the ability of 3D cultured JPCs to modulate macrophage plasticity.

Mechanical and Functional Improvement of β-TCP Scaffolds for Use in Bone Tissue Engineering.

Autologous bone transplantation is still considered as the gold standard therapeutic option for bone defect repair. The alternative tissue engineering approaches have to combine good hardiness of biomaterials whilst allowing good stem cell functionality. To become more useful for load-bearing applications, mechanical properties of calcium phosphate materials have to be improved. In the present study, we aimed to reduce the brittleness of β-tricalcium phosphate (β-TCP). For this purpose, we used three polymers (PDL-02, -02a, -04) for coatings and compared resulting mechanical and degradation properties as well as their impact on seeded periosteal stem cells. Mechanical properties of coated and uncoated β-TCP scaffolds were analyzed. In addition, degradation kinetics analyses of the polymers employed and of the polymer-coated scaffolds were performed. For bioactivity assessment, the scaffolds were seeded with jaw periosteal cells (JPCs) and cultured under untreated and osteogenic conditions. JPC adhesion/proliferation, gene and protein expression by immunofluorescent staining of embedded scaffolds were analyzed. Raman spectroscopy measurements gave an insight into material properties and cell mineralization. PDL-coated β-TCP scaffolds showed a significantly higher flexural strength in comparison to that of uncoated scaffolds. Degradation kinetics showed considerable differences in pH and electrical conductivity of the three different polymer types, while the core material β-TCP was able to stabilize pH and conductivity. Material differences seemed to have an impact on JPC proliferation and differentiation potential, as reflected by the expression of osteogenic marker genes. A homogenous cell colonialization of coated and uncoated scaffolds was detected. Most interesting from a bone engineer's point of view, the PDL-04 coating enabled detection of cell matrix mineralization by Raman spectroscopy. This was not feasible with uncoated scaffolds, due to intercalating effects of the β-TCP material and the JPC-formed calcium phosphate. In conclusion, the use of PDL-04 coating improved the mechanical properties of the β-TCP scaffold and promoted cell adhesion and osteogenic differentiation, whilst allowing detection of cell mineralization within the ceramic core material.

Secretomes derived from osteogenically differentiated jaw periosteal cells inhibit phenotypic and functional maturation of CD14+ monocyte-derived dendritic cells.

The jaw periosteal tissue is generally recognized as a suitable source for the isolation of mesenchymal stem cells (MSCs). In previous studies we showed evidence that two- and three-dimensionally cultured jaw periosteum-derived MSCs (JPCs) are able to induce a more immature phenotype of dendritic cells (DCs). To further expand our knowledge of JPCs' immunoregulative function, we investigated the effects of JPC secretomes derived from undifferentiated (CO) or osteogenically differentiated cells (treated with or without dexamethasone: OB+/-D) on CD14+ monocyte-derived DCs (MoDCs). We detected a remarkably reduced formation of MoDC homotypic clusters under the influence of secretomes from osteogenically induced JPCs. Further, significantly decreased numbers of CD83+ cells, up-regulated CD209 and down-regulated CD80, CD86 and CD197 expression levels were detected on the surface of MoDCs. Whereas secretomes from JPCs osteogenically stimulated with dexamethasone significantly enhanced FITC-dextran uptake capacity of MoDCs, the increase by secretomes of JPCs treated without dexamethasone did not reach significance. The analysis of mixed lymphocyte reactions revealed that OB+/-D secretomes were able to significantly reduce the numbers of proliferating CD14- peripheral blood mononuclear cells (PBMCs) and of proliferating CD4+ T cells. The OB-D secretome significantly promoted the expansion of regulatory CD25+ T cells. Regarding gene expression of MoDCs, remarkably up-regulated mRNA expression of CD209, HLA-DRA, CSF3, IL10 and IL8 was detected when DCs were cultured in the presence of OB+/-D secretomes. At the same time, secretomes seemed to have an impact in the down-regulation of IFNγ and IL12B gene expression. At protein level, OB+/-D secretomes significantly up-regulated IL-10 and IDO (indoleamine-pyrrole 2,3-dioxygenase) levels whereas IL-12/IL-23p40 levels were down-regulated in supernatants of MoDCs when cultured under the presence of OB+/-D secretomes. Taken together, while secretomes from untreated JPCs had only little effects on the process of maturation of MoDCs, secretomes derived from osteogenically induced JPCs were able to inhibit the phenotypic and functional maturation of MoDCs.

How osteogenic is dexamethasone?-effect of the corticosteroid on the osteogenesis, extracellular matrix, and secretion of osteoclastogenic factors of jaw periosteum-derived mesenchymal stem/stromal cells.

Dexamethasone (dexa) is commonly used to stimulate osteogenic differentiation of mesenchymal stem/stromal cells (MSCs) in vitro. However, it is paradoxical that glucocorticoids (GCs) such as dexa lead to bone loss and increased fracture risk in patients undergoing glucocorticoid therapy, causing glucocorticoid-induced osteoporosis (GIOP). In a recent publication, we demonstrated that osteogenic differentiation of progenitor cells isolated from jaw periosteal tissue (JPCs) does not depend on dexa, if the medium is supplemented with human platelet lysate (hPL) instead of fetal bovine serum (FBS). This allows the in vitro conditions to be much closer to the natural situation in vivo and enables us to compare osteogenic differentiation with and without dexa. In the present study, we demonstrate that the absence of dexa did not reduce mineralization capacity, but instead slightly improved the osteogenic differentiation of jaw periosteal cells. On the other hand, we show that dexa supplementation strongly alters the gene expression, extracellular matrix (ECM), and cellular communication of jaw periosteal cells. The secretome of periosteal cells previously treated with an osteogenic medium with and without dexa was used to investigate the changes in paracrine secretion caused by dexa. Dexa altered the secretion of several cytokines by jaw periosteal cells and strongly induced osteoclast differentiation of peripheral blood mononuclear cells (PBMCs). This study demonstrates how dexa supplementation can influence the outcome of in vitro studies and highlights a possible role of periosteal cells in the pathogenesis of glucocorticoid-induced osteoporosis. The methods used here can serve as a model for studying bone formation, fracture healing, and various pathological conditions such as (glucocorticoid-induced) osteoporosis, osteoarthritis, bone cancer, and others, in which the interactions of osteoblasts with surrounding cells play a key role.

Impact of Fluid Dynamics on the Viability and Differentiation Capacity of 3D-Cultured Jaw Periosteal Cells

Perfused bioreactor systems are considered to be a promising approach for the 3D culturing of stem cells by improving the quality of the tissue-engineered grafts in terms of better cell proliferation and deeper penetration of used scaffold materials. Our study aims to establish an optimal perfusion culture system for jaw periosteal cell (JPC)-seeded scaffolds. For this purpose, we used beta-tricalcium phosphate (β-TCP) scaffolds as a three-dimensional structure for cell growth and osteogenic differentiation. Experimental set-ups of tangential and sigmoidal fluid configurations with medium flow rates of 100 and 200 µL/min were applied within the perfusion system. Cell metabolic activities of 3D-cultured JPCs under dynamic conditions with flow rates of 100 and 200 µL/min were increased in the tendency after 1, and 3 days of culture, and were significantly increased after 5 days. Significantly higher cell densities were detected under the four perfused conditions compared to the static condition at day 5. However, cell metabolic and proliferation activity under dynamic conditions showed flow rate independency in our study. In this study, dynamic conditions increased the expression of osteogenic markers (ALPL, COL1A1, RUNX2, and OCN) compared to static conditions and the tangential configuration showed a stronger osteogenic effect than the sigmoidal flow configuration.

Influence of Human Jaw Periosteal Cells Seeded β-Tricalcium Phosphate Scaffolds on Blood Coagulation.

Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated β-tricalcium phosphate (β-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated β-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated β-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (β-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded β-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.

Jaw Periosteum-Derived Mesenchymal Stem Cells Regulate THP-1-Derived Macrophage Polarization.

Mesenchymal stem cells from bone marrow have powerful immunomodulatory capabilities. The interactions between jaw periosteal cells (JPCs) and macrophages are not only relevant for the application of JPCs in regenerative medicine, but this understanding could also help treating diseases like osteonecrosis of the jaw. In previous studies, we analyzed, for the first time, immunomodulatory features of 2D- and 3D-cultured JPCs. In the present work, the effects of JPCs on the polarization state of macrophages in contact coculture were analyzed. To improve the macrophage polarization study, different concentrations of PMA (5 nM, 25 nM, and 150 nM) or different medium supplementations (10% FBS, 10% hPL and 5% hPL) were compared. Further, in order to analyze the effects of JPCs on macrophage polarization, JPCs and PMA-stimulated THP-1 cells were cocultured under LPS/IFN-γ or IL-4/IL-13 stimulatory conditions. Surface marker expression of M1 and M2 macrophages were analyzed under the different culture supplementations in order to investigate the immunomodulatory properties of JPCs. Our results showed that 5 nM PMA can conduct an effective macrophage polarization. The analyses of morphological parameters and surface marker expression showed more distinct M1/M2 phenotypes over FBS supplementation when using 5% hPL during macrophage polarization. In the coculture, immunomodulatory properties of JPCs improved significantly under 5% hPL supplementation compared to other supplementations. We concluded that, under the culture condition with 5% hPL, JPCs were able to effectively induce THP-1-derived macrophage polarization.

An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs.

The generation of autologous human induced pluripotent stem cells (hiPSCs) from a patient’s somatic cells and the sub­sequent differentiation of these cells into desired cell types offer innovative treatment options for tissue regeneration. The hiPSCs obtained are usually implanted in immunodeficient mice, and teratoma formation is analyzed after 4 to 6 weeks to assess the cells’ pluripotency. In this study, an alternative in vivo model based on chicken egg chorioallantoic membrane (CAM) was established to analyze the pluripotency of newly created hiPSCs. 0.5, 1, 2, 4 x 106 hiPSCs gen­erated from urine-derived renal epithelial cells were seeded on CAM and incubated for 9 days. Teratoma formation was detected in 70% of eggs inoculated with 2 x 106 hiPSCs and in 100% of eggs inoculated with 4 x 106 hiPSCs. All teratomas exhibited vascular structures. The robustness of the CAM model was confirmed using two additional hiPSC lines derived from human fibroblasts (NuFFs) or jaw periosteal cells. The presence of all three germ layers within the teratomas was successfully verified by histochemical and immunofluorescence staining and gene expression analysis of germ layer-specific markers. Urine-derived renal epithelial cells were used as negative control and showed no teratoma formation. The CAM-based in vivo model provides an optimal in vivo test environment for the pluripotency evaluation of newly generated hiPSC lines. This simple, fast, inexpensive and reproducible method reduces the suffering of animals and thus implements the principles of the 3Rs (replacement, reduction, and refinement).

An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs_suppl

The generation of autologous human induced pluripotent stem cells (hiPSCs) from a patient’s somatic cells and the sub­sequent differentiation of these cells into desired cell types offer innovative treatment options for tissue regeneration. The hiPSCs obtained are usually implanted in immunodeficient mice, and teratoma formation is analyzed after 4 to 6 weeks to assess the cells’ pluripotency. In this study, an alternative in vivo model based on chicken egg chorioallantoic membrane (CAM) was established to analyze the pluripotency of newly created hiPSCs. 0.5, 1, 2, 4 x 106 hiPSCs gen­erated from urine-derived renal epithelial cells were seeded on CAM and incubated for 9 days. Teratoma formation was detected in 70% of eggs inoculated with 2 x 106 hiPSCs and in 100% of eggs inoculated with 4 x 106 hiPSCs. All teratomas exhibited vascular structures. The robustness of the CAM model was confirmed using two additional hiPSC lines derived from human fibroblasts (NuFFs) or jaw periosteal cells. The presence of all three germ layers within the teratomas was successfully verified by histochemical and immunofluorescence staining and gene expression analysis of germ layer-specific markers. Urine-derived renal epithelial cells were used as negative control and showed no teratoma formation. The CAM-based in vivo model provides an optimal in vivo test environment for the pluripotency evaluation of newly generated hiPSC lines. This simple, fast, inexpensive and reproducible method reduces the suffering of animals and thus implements the principles of the 3Rs (replacement, reduction, and refinement).

Jaw Periosteal Cells Seeded in Beta-Tricalcium Phosphate Inhibit Dendritic Cell Maturation.

Mesenchymal stem cells (MSCs) have gained attraction not only in the field of regenerative medicine but also in the field of autoimmune disease therapies or organ transplantation due to their immunoregulatory and/or immunosuppressive features. Dendritic cells (DCs) play a crucial role in initiating and regulating immune reactions by promoting antigen-specific T cell activation. In this study, we investigated the effect of human jaw periosteal progenitor cells (JPCs) seeded in beta-tricalcium phosphate (β-TCP) scaffolds on monocyte-derived DC differentiation. Significantly lower numbers of differentiated DCs were observed in the presence of normal (Co) and osteogenically induced (Ob) JPCs-seeded β-TCP constructs. Gene expression analysis revealed significantly lower interleukin-12 subunit p35 (IL-12p35) and interleukin-12 receptor beta 2 (IL-12Rβ2) and pro-inflammatory cytokine interferon-gamma (IFN-γ) levels in DCs under Ob conditions, while interleukin-8 (IL-8) gene levels were significantly increased. Furthermore, in the presence of JPCs-seeded β-TCP constructs, interleukin-10 (IL-10) gene expression was significantly induced in DCs, particularly under Ob conditions. Analysis of DC protein levels shows that granulocyte-colony stimulating factor (G-CSF) was significantly upregulated in coculture groups. Our results indicate that undifferentiated and osteogenically induced JPCs-seeded β-TCP constructs have an overall inhibitory effect on monocyte-derived DC maturation.

Establishment of Collagen: Hydroxyapatite/BMP-2 Mimetic Peptide Composites.

Extensive efforts were undertaken to develop suitable biomaterials for tissue engineering (TE) applications. To facilitate clinical approval processes and ensure the success of TE applications, bioinspired concepts are currently focused on. Working on bone tissue engineering, we describe in the present study a method for biofunctionalization of collagen/hydroxyapatite composites with BMP-2 mimetic peptides. This approach is expected to be fundamentally transferable to other tissue engineering fields. A modified BMP-2 mimetic peptide containing a negatively charged poly-glutamic acid residue (E7 BMP-2 peptide) was used to bind positively charged hydroxyapatite (HA) particles by electrostatic attraction. Binding efficiency was biochemically detected to be on average 85% compared to 30% of BMP-2 peptide without E7 residue. By quartz crystal microbalance (QCM) analysis, we could demonstrate the time-dependent dissociation of the BMP-2 mimetic peptides and the stable binding of the E7 BMP-2 peptides on HA-coated quartz crystals. As shown by immunofluorescence staining, alkaline phosphatase expression is similar to that detected in jaw periosteal cells (JPCs) stimulated with the whole BMP-2 protein. Mineralization potential of JPCs in the presence of BMP-2 mimetic peptides was also shown to be at least similar or significantly higher when low peptide concentrations were used, as compared to JPCs cultured in the presence of recombinant BMP-2 controls. In the following, collagen/hydroxyapatite composite materials were prepared. By proliferation analysis, we detected a decrease in cell viability with increasing HA ratios. Therefore, we chose a collagen/hydroxyapatite ratio of 1:2, similar to the natural composition of bone. The following inclusion of E7 BMP-2 peptides within the composite material resulted in significantly elevated long-term JPC proliferation under osteogenic conditions. We conclude that our advanced approach for fast and cost-effective scaffold preparation and biofunctionalization is suitable for improved and prolonged JPC proliferation. Further studies should prove the functionality of composite scaffolds in vivo.

IPSC-Derived MSCs Versus Originating Jaw Periosteal Cells: Comparison of Resulting Phenotype and Stem Cell Potential.

Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-β-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells.

Quality Analysis of Minerals Formed by Jaw Periosteal Cells under Different Culture Conditions.

Previously, we detected a higher degree of mineralization in fetal calf serum (FCS) compared to serum-free cultured jaw periosteum derived osteoprogenitor cells (JPCs). By Raman spectroscopy, we detected an earlier formation of mineralized extracellular matrix (ECM) of higher quality under serum-free media conditions. However, mineralization potential remained too low. In the present study, we aimed to investigate the biochemical composition and subsequent biomechanical properties of the JPC-formed ECM and minerals under human platelet lysate (hPL) and FCS supplementation. JPCs were isolated (n = 4 donors) and expanded under FCS conditions and used in passage five for osteogenic induction under both, FCS and hPL media supplementation. Raman spectroscopy and Alizarin Red/von Kossa staining were employed for biochemical composition analyses and for visualization and quantification of mineralization. Osteocalcin gene expression was analyzed by quantitative PCR. Biomechanical properties were assessed by using atomic force microscopy (AFM). Raman spectroscopic measurements showed significantly higher (p < 0.001) phosphate to protein ratios and in the tendency, lower carbonate to phosphate ratios in osteogenically induced JPCs under hPL in comparison to FCS culturing. Furthermore, higher crystal sizes were detected under hPL culturing of the cells. With respect to the ECM, significantly higher ratios of the precursor protein proline to hydroxyproline were detected in hPL-cultured JPC monolayers (p < 0.001). Additionally, significantly higher levels (p < 0.001) of collagen cross-linking were calculated, indicating a higher degree of collagen maturation in hPL-cultured JPCs. By atomic force microscopy, a significant increase in ECM stiffness (p < 0.001) of FCS cultured JPC monolayers was observed. The reverse effect was measured for the JPC formed precipitates/minerals. Under hPL supplementation, JPCs formed minerals of significantly higher stiffness (p < 0.001) when compared to the FCS setting. This study demonstrates that hPL culturing of JPCs leads to the formation of an anorganic material of superior quality in terms of biochemical composition and mechanical properties.

Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA.

Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.

Effects of Jaw Periosteal Cells on Dendritic Cell Maturation.

Clinical application of tissue engineering products requires the exclusion of immune responses after implantation. We used jaw periosteal cells (JPCs) as a suitable stem cell source and analyzed herein the effects of JPCs on dendritic cell maturation after co-culturing of both cell types. Peripheral blood mononuclear cells (PBMCs) were differentiated to dendritic cells (DCs) by the addition of differentiation cocktails for 7 days in co-culture with undifferentiated and osteogenically induced JPCs. The effects of JPCs on DC maturation were analyzed at the beginning (day 7), in the middle (day 14), and at the end (day 21) of the osteogenesis process. We detected significantly lower DC numbers after co-culturing with JPCs that have previously been left untreated or osteogenically differentiated for 7, 14, and 21 days. Using gene expression analyses, significantly lower IL-12p35 and -p40 and pro-inflammatory cytokine (IFN-γ and TNF-α) levels were detected, whereas IL-8 mRNA levels were significantly higher in DCs. Furthermore, osteogenic media conditions enhanced significantly IL-10 gene expression. We concluded that undifferentiated and osteogenically differentiated JPCs had an overall inhibiting influence on dendritic cell maturation. Further studies should clarify the underlaying mechanism in depth.

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization.

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

Raman Spectroscopic Analyses of Jaw Periosteal Cell Mineralization.

To achieve safer patient treatments, serum-free cell culture conditions have to be established for cell therapies. In previous studies, we demonstrated that serum-free culture favored the proliferation of MSCA-1+ osteoprogenitors derived from the jaw periosteum. In this study, the in vitro formation of bone-specific matrix by MSCA-1+ jaw periosteal cells (JPCs, 3 donors) was assessed and compared under serum-free and serum-containing media conditions using the marker-free Raman spectroscopy. Based on a standard fluorescence assay, JPCs from one patient were not able to mineralize under serum-containing culture conditions, whereas the other cells showed similar mineralization levels under both conditions. Raman spectra from mineralizing MSCA-1+ JPCs revealed higher levels of hydroxyapatite formation and higher mineral to matrix ratios under serum-free culture conditions. Higher carbonate to phosphate ratios and higher crystallinity in JPCs cultured under serum-containing conditions indicated immature bone formation. Due to reduced collagen production under serum-free conditions, we obtained significant differences in collagen maturity and proline to hydroxyproline ratios compared to serum-free conditions. We conclude that Raman spectroscopy is a useful tool for the assessment and noninvasive monitoring of in vitro mineralization of osteoprogenitor cells. Further studies should extend this knowledge and improve JPC mineralization by optimizing culture conditions.

Surface biofunctionalization of β-TCP blocks using aptamer 74 for bone tissue engineering

Successful bone regeneration following oral and maxillofacial surgeries depends on efficient functionalization strategies that allow the recruitment of osteogenic progenitor cells at the tissue/implant interface. We have previously identified aptamer 74, which exhibited a binding affinity for osteogenically induced jaw periosteal cells (JPCs). In the present study, this aptamer was used for the surface biofunctionalization of β-tricalcium phosphate (β-TCP) blocks.Atomic force microscopy (AFM) measurements showed increased binding activity of aptamer 74 towards osteogenically induced JPCs compared to untreated controls. The immobilization efficiency of aptamer 74 was analyzed using the QuantiFluor ssDNA assay for 2D surfaces and by amino acid analysis for 3D β-TCP constructs. Following the successful immobilization of aptamer 74 in 2D culture wells and on 3D constructs, in vitro assays showed no significant differences in cell proliferation compared to unmodified surfaces. Interestingly, JPC mineralization was significantly higher on the 2D surfaces and higher cell adhesion was detected on the 3D constructs with immobilized aptamer.Herein, we report an established, biocompatible β-TCP matrix with surface immobilization of aptamer 74, which enhances properties such as cell adhesion on 3D constructs and mineralization on 2D surfaces. Further studies need to be performed to improve the immobilization efficiency and to develop a suitable approach for JPC mineralization growing within 3D β-TCP constructs.

Coating of ß-tricalcium phosphate scaffolds—a comparison between graphene oxide and poly-lactic-co-glycolic acid

Bone regeneration in critical size defects is a major challenge in oral and maxillofacial surgery, and the gold standard for bone reconstruction still requires the use of autologous tissue. To overcome the need for a second intervention and to minimize morbidity, the development of new biomaterials with osteoinductive features is the focus of current research. As a scaffolding material, ß-tricalcium phosphate (ß-TCP) is suitable for bone regeneration purposes, although it does not carry any functional groups for the covalent immobilization of molecules.The aim of the present study was to establish effective coating variants for ß-TCP constructs to enable the biofunctionalization of anorganic blocks with different osteogenic molecules in future studies. We established working protocols for thin surface coatings consisting of polylactic-co-glycolic acid (PLGA) and graphene oxide (GO) by varying parameters. Surface properties such as the angularity and topography of the developed scaffolds were analyzed. To examine biological functionality, the adhesion and proliferation behavior of jaw periosteal cells (JPCs) were tested on the coated constructs.Our results suggest that PLGA is the superior material for surface coating of ß-TCP matrices, leading to higher JPC proliferation rates and providing a more suitable basis for further biofunctionalization in the field of bone tissue engineering.

Selection of Osteoprogenitors from the Jaw Periosteum by a Specific Animal-Free Culture Medium

The goal of our research work is to establish mesenchymal osteoprogenitors derived from human jaw periosteum for tissue engineering applications in oral and maxillofacial surgery. For future autologous and/or allogeneic transplantations, some issues must be addressed. On the one hand, animal-free culture conditions have yet to be established. On the other hand, attempts should be undertaken to shorten the in vitro culturing process efficiently. The aim of the present study is to compare and analyze the phenotype of osteoprogenitors from the jaw periosteum under normal FCS-containing and animal-free culture conditions. Therefore, we analyzed the proliferation rates of MesenCult-XF medium (MC-) in comparison to DMEM-cultured JPCs. Whereas jaw periosteal cells (JPCs) show relatively slow proliferation rates and a fibroblastoid shape under DMEM culture conditions, MC-cultured JPCs diminished their cell size significantly and proliferated rapidly. By live-monitoring measurements of adhesion and proliferation, we made an interesting observation: whereas the proliferation of the MSCA-1+ subpopulation and the unseparated cell fraction were favored by the animal-free culture medium, the proliferation of the MSCA-1- subpopulation seemed to be repressed under these conditions. The alkaline phosphatase expression pattern showed similar results under both culture conditions. Comparison of the mineralization capacity revealed an earlier formation of calcium-phosphate precipitates under MC culture conditions; however, the mineralization capacity of the DMEM-cultured cells seemed to be higher. We conclude that the tested animal-free medium is suitable for the in vitro expansion and even for the specific selection of osteoprogenitor cells derived from the jaw periosteum.