[Isolation and characterization of normal mandibular periosteal stem cells from human and macaca mulatta and cross-species single-cell analysis].

Objective: To investigate the presence of a distinct stem cell populations different from mesenchymal stem cells in the mandibular periosteum of both human and non-human primates (macaca mulatta), to explore its properties during intramembranous osteogenesis and to establish standard protocols for the isolation, culturing and expanding of mandibular periosteal stem cells (PSC) distinguished from other PSCs in other anatomical regions. Methods: Periosteum was harvested from the bone surface during flap bone removal in patients aged 18-24 years undergoing third molar extraction and from the buccal side of the mandibular premolar region of 6-year-old macaca mulatta respectively, and then subjected to single-cell sequencing using the Illumina platform Novaseq 6000 sequencer. Cross-species single-cell transcriptome sequencing results were compared using homologous gene matching. PSC were isolated from primary tissues using two digestion methods with body temperature and low temperature, and their surface markers (CD200, CD31, CD45 and CD90) were identified by cell flow cytometry. The ability of cell proliferation and three-lineage differentiation of PSC expanded to the third generation in vitro in different species were evaluated. Finally, the similarities and differences in osteogenic properties of PSC and bone marrow mesenchymal stem cells (BMSC) were compared. Results: The single-cell sequencing results indicated that 18 clusters of cell populations were identified after homologous gene matching for dimensionality reduction, and manual cellular annotation was conducted for each cluster based on cell marker databases. The comparison of different digestion protocols proved that the low-temperature overnight digestion protocol can stably isolate PSC from the human and m. mulatta mandibular periosteum and the cells exhibited a fibroblast-like morphology. This research confirmed that PSC of human and m. mulatta had similar proliferation capabilities through the cell counting kit-8 assay. Flow cytometry analysis was then used to identify the cells isolated from the periosteum expressed CD200(+), CD31(-), CD45(-), CD90(-). Then, human and m. mulatta PSC were induced into osteogenesis, adipogenesis, and chondrogenesis to demonstrate their corresponding multi-lineage differentiation capabilities. Finally, comparison with BMSC further clarified the oesteogenesis characteristics of PSC. The above experiments proved that the cells isolated from the periosteum were peiosteal cells with characteristics of stem cells evidenced by their cell morphology, proliferation ability, surface markers, and differentiation ability, and that this group of PSC possessed characteristics different from traditional mesenchymal stem cells. Conclusions: In this study, normal mandibular PSC from humans and m. mulatta were stably isolated and identified for the first time, providing a cellular foundation for investigating the mechanism of mandibular intramembranous osteogenesis, exploring ideal non-human primate models and establishing innovative strategies for clinically mandibular injury repair.

Endothelial to mesenchymal Notch signaling regulates skeletal repair.

We present a transcriptomic analysis that provides a better understanding of regulatory mechanisms within the healthy and injured periosteum. The focus of this work is on characterizing early events controlling bone healing during formation of periosteal callus on day 3 after fracture. Building on our previous findings showing that induced Notch1 signaling in osteoprogenitors leads to better healing, we compared samples in which the Notch 1 intracellular domain is overexpressed by periosteal stem/progenitor cells, with control intact and fractured periosteum. Molecular mechanisms and changes in skeletal stem/progenitor cells (SSPCs) and other cell populations within the callus, including hematopoietic lineages, were determined. Notably, Notch ligands were differentially expressed in endothelial and mesenchymal populations, with Dll4 restricted to endothelial cells, whereas Jag1 was expressed by mesenchymal populations. Targeted deletion of Dll4 in endothelial cells using Cdh5CreER resulted in negative effects on early fracture healing, while deletion in SSPCs using α-smooth muscle actin-CreER did not impact bone healing. Translating these observations into a clinically relevant model of bone healing revealed the beneficial effects of delivering Notch ligands alongside the osteogenic inducer, BMP2. These findings provide insights into the regulatory mechanisms within the healthy and injured periosteum, paving the way for novel translational approaches to bone healing.

Vitamin A enhanced periosteal osteoclastogenesis is associated with increased number of tissue-derived macrophages/osteoclast progenitors.

A deleterious effect of elevated levels of vitamin A on bone health has been reported in clinical studies. Mechanistic studies in rodents have shown that numbers of periosteal osteoclasts are increased, while endocortical osteoclasts are simultaneously decreased by vitamin A treatment. The present study investigated the in vitro and in vivo effect of all-trans retinoic acid (ATRA), the active metabolite of vitamin A, on periosteal osteoclast progenitors. Mouse calvarial bone cells were cultured in media containing ATRA, with or without the osteoclastogenic cytokine receptor activator of nuclear factor kappa B-ligand (RANKL), on plastic dishes or bone discs. Whereas ATRA did not stimulate osteoclast formation alone, the compound robustly potentiated the formation of RANKL-induced bone resorbing osteoclasts. This effect was due to stimulation by ATRA (half-maximal stimulation ∼3 nM) on the numbers of macrophages/osteoclast progenitors in the bone cell cultures, as assessed by mRNA and protein expression of several macrophage and osteoclast progenitor cell markers, such as macrophage colony-stimulating factor receptor, receptor activator of nuclear factor kappa B, F4/80, and CD11b, as well as by flow cytometry (FACS) analysis of CD11b+/F480+/Gr1- cells. The stimulation of macrophage numbers in the periosteal cell cultures was not mediated by increased macrophage colony-stimulating factor or interleukin-34. In contrast, ATRA did not enhance macrophages in bone marrow cell cultures. Importantly, ATRA treatment upregulated the mRNA expression of several macrophage-related genes in the periosteum of tibia in adult mice. These observations demonstrate a novel mechanism by which vitamin A enhances osteoclast formation specifically on periosteal surfaces.

Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone.

Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.

Isolation, identification and osteogenic capability analysis of mesenchymal stem cells derived from different layers of human maxillary sinus membrane.

AIM: To discover the populations of mesenchymal stem cells (MSCs) derived from different layers of human maxillary sinus membrane (hMSM) and evaluate their osteogenic capability. MATERIALS AND METHODS: hMSM was isolated into a monolayer using the combined method of physical separation and enzymatic digestion. The localization of MSCs in hMSM was performed by immunohistological staining and other techniques. Lamina propria layer-derived MSCs (LMSCs) and periosteum layer-derived MSCs (PMSCs) from hMSM were expanded using the explant cell culture method and identified by multilineage differentiation assays, colony formation assay, flow cytometry and so on. The biological characteristics of LMSCs and PMSCs were compared using RNA sequencing, reverse transcription and quantitative polymerase chain reaction, immunofluorescence staining, transwell assay, western blotting and so forth. RESULTS: LMSCs and PMSCs from hMSMs were both CD73-, CD90- and CD105-positive, and CD34-, CD45- and HLA-DR-negative. LMSCs and PMSCs were identified as CD171+/CD90+ and CD171-/CD90+, respectively. LMSCs displayed stronger proliferation capability than PMSCs, and PMSCs presented stronger osteogenic differentiation capability than LMSCs. Moreover, PMSCs could recruit and promote osteogenic differentiation of LMSCs. CONCLUSIONS: This study identified and isolated two different types of MSCs from hMSMs. Both MSCs served as good potential candidates for bone regeneration.

Discovery of CTSK+ Periosteal Stem Cells Mediating Bone Repair in Orbital Reconstruction.

Purpose: The purpose of this study was to explore the role of cathepsin K positive (CTSK+) periosteal stem cells (PSCs) in orbital bone repair and to clarify the source of endogenous stem cells for orbital bone self-repair. Methods: Periosteum samples obtained by clinical orbital bone repair surgery were analyzed, after which immunofluorescence and immunohistochemical staining were used to detect the content of bone marrow-derived cells and CTSK+ PSCs in periosteum as well as the mobilization of PSCs. CTSK+ PSCs were characterized by flow cytometry. Transcriptome sequencing was used to compare the transcriptomic characteristics of CTSK+ PSCs and bone marrow mesenchymal stem cells (BMSCs). Results: The orbital periosteum contained CTSK+CD200+ cell lineage, including CD200+CD105- PSCs and CD200+CD105+ progenitor cells. CTSK and osteocalcin (OCN) colocalized in the inner layer of the orbital periosteum, suggesting the osteogenic differentiation potential of CTSK+ PSCs. CTSK expression was much higher in periosteum after mobilization. Immunofluorescence showed low amounts of scattered CD31+ and CD45+ cells in the orbital periosteum. The stem cell characteristics of CTSK+ PSCs were verified by multidirectional differentiation. Flow cytometry found CD200+CD105- CTSK+ PSCs and CD200variantCD105+ progenitor cells. Transcriptome sequencing of CTSK+ PSCs and BMSCs found 3613 differential genes with significant differences. Gene Ontology (GO) analysis showed the differences between the two types of stem cells, revealing that PSCs were more suitable for intramembranous osteogenesis. Conclusions: CTSK+ PSCs may be endogenous stem cells for orbital bone repair. They are mobilized after orbital fracture and have unique features suitable for intramembranous osteogenesis, completely different from BMSCs.

Mechanical Properties of Human Concentrated Growth Factor (CGF) Membrane and the CGF Graft with Bone Morphogenetic Protein-2 (BMP-2) onto Periosteum of the Skull of Nude Mice.

Concentrated growth factor (CGF) is 100% blood-derived, cross-linked fibrin glue with platelets and growth factors. Human CGF clot is transformed into membrane by a compression device, which has been widely used clinically. However, the mechanical properties of the CGF membranes have not been well characterized. The aims of this study were to measure the tensile strength of human CGF membrane and observe its behavior as a scaffold of BMP-2 in ectopic site over the skull. The tensile test of the full length was performed at the speed of 2mm/min. The CGF membrane (5 × 5 × 2 mm3) or the CGF/BMP-2 (1.0 µg) membrane was grafted onto the skull periosteum of nude mice (5-week-old, male), and harvested at 14 days after the graft. The appearance and size of the CGF membranes were almost same for 7 days by soaking at 4 °C in saline. The average values of the tensile strength at 0 day and 7 days were 0.24 MPa and 0.26 MPa, respectively. No significant differences of both the tensile strength and the elastic modulus were found among 0, 1, 3, and 7 days. Supra-periosteal bone induction was found at 14 days in the CGF/BMP-2, while the CGF alone did not induce bone. These results demonstrated that human CGF membrane could become a short-term, sticky fibrin scaffold for BMP-2, and might be preserved as auto-membranes for wound protection after the surgery.

Influence of Human Jaw Periosteal Cells Seeded ß-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 ß-tricalcium phosphate (ß-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 ß-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 ß-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 (ß-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 ß-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.

Isolation and Culture of Periosteum-Derived Progenitor Cells from Mice.

This chapter describes the methods of isolation of mouse periosteal progenitor cells. There are three basic methods utilized. The bone grafting method was developed utilizing the fracture healing process to expand the progenitor populations. Bone capping methods requires enzymatic digestion and purification of cells from the native periosteum, while the Egression/Explant method requires the least manipulation with placement of cortical bone fragments with attached periosteum in a culture dish. Various cell surface antibodies have been employed over the years to characterize periosteum derived progenitor cells, but the most consistent minimal criteria was recommended by the International Society for Cellular Therapy. Confirmation of the multipotent status of these isolated cells can be achieved by differentiation into the three basic mesodermal lineages in vitro.

Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts.

Chondrocytes proliferate and mature into hypertrophic chondrocytes. Vascular invasion into the cartilage occurs in the terminal hypertrophic chondrocyte layer, and terminal hypertrophic chondrocytes die by apoptosis or transdifferentiate into osteoblasts. Runx2 is essential for osteoblast differentiation and chondrocyte maturation. Runx2-deficient mice are composed of cartilaginous skeletons and lack the vascular invasion into the cartilage. However, the requirement of Runx2 in the vascular invasion into the cartilage, mechanism of chondrocyte transdifferentiation to osteoblasts, and its significance in bone development remain to be elucidated. To investigate these points, we generated Runx2fl/flCre mice, in which Runx2 was deleted in hypertrophic chondrocytes using Col10a1 Cre. Vascular invasion into the cartilage was similarly observed in Runx2fl/fl and Runx2fl/flCre mice. Vegfa expression was reduced in the terminal hypertrophic chondrocytes in Runx2fl/flCre mice, but Vegfa was strongly expressed in osteoblasts in the bone collar, suggesting that Vegfa expression in bone collar osteoblasts is sufficient for vascular invasion into the cartilage. The apoptosis of terminal hypertrophic chondrocytes was increased and their transdifferentiation was interrupted in Runx2fl/flCre mice, leading to lack of primary spongiosa and osteoblasts in the region at E16.5. The osteoblasts appeared in this region at E17.5 in the absence of transdifferentiation, and the number of osteoblasts and the formation of primary spongiosa, but not secondary spongiosa, reached to levels similar those in Runx2fl/fl mice at birth. The bone structure and volume and all bone histomophometric parameters were similar between Runx2fl/fl and Runx2fl/flCre mice after 6 weeks of age. These findings indicate that Runx2 expression in terminal hypertrophic chondrocytes is not required for vascular invasion into the cartilage, but is for their survival and transdifferentiation into osteoblasts, and that the transdifferentiation is necessary for trabecular bone formation in embryonic and neonatal stages, but not for acquiring normal bone structure and volume in young and adult mice.

TGF-ß/Smad2 signalling regulates enchondral bone formation of Gli1+ periosteal cells during fracture healing.

OBJECTIVES: Most bone fracture heals through enchondral bone formation that relies on the involvement of periosteal progenitor cells. However, the identity of periosteal progenitor cells and the regulatory mechanism of their proliferation and differentiation remain unclear. The aim of this study was to investigate whether Gli1-CreERT2 can identify a population of murine periosteal progenitor cells and the role of TGF-ß signalling in periosteal progenitor cells on fracture healing. MATERIALS AND METHODS: Double heterozygous Gli1-CreERT2 ;Rosa26-tdTomatoflox/wt mice were sacrificed at different time points for tracing the fate of Gli1+ cells in both intact and fracture bone. Gli1-CreERT2 -mediated Tgfbr2 knockout (Gli1-CreERT2 ;Tgfbr2flox/flox ) mice were subjected to fracture surgery. At 4, 7, 10, 14 and 21 days post-surgery, tibia samples were harvested for tissue analyses including µCT, histology, real-time PCR and immunofluorescence staining. RESULTS: Through cell lineage-tracing experiments, we have revealed that Gli1-CreER T2 can be used to identify a subpopulation of periosteal progenitor cells in vivo that persistently reside in periosteum and contribute to osteochondral elements during fracture repair. During the healing process, TGF-ß signalling is continually activated in the reparative Gli1+ periosteal cells. Conditional knockout of Tgfbr2 in these cells leads to a delayed and impaired enchondral bone formation, at least partially due to the reduced proliferation and chondrogenic and osteogenic differentiation of Gli1+ periosteal cells. CONCLUSIONS: TGF-ß signalling plays an essential role on fracture repair via regulating enchondral bone formation process of Gli1+ periosteal cells.

Resveratrol can enhance osteogenic differentiation and mitochondrial biogenesis from human periosteum-derived mesenchymal stem cells.

BACKGROUND: Osteoporosis is a metabolic bone disorder that leads to low bone mass and microstructural deterioration of bone tissue and increases bone fractures. Resveratrol, a natural polyphenol compound, has pleiotropic effects including anti-oxidative, anti-aging, and anti-cancer effects. Resveratrol also has roles in increasing osteogenesis and in upregulating mitochondrial biogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is still unclear that resveratrol can enhance osteogenic differentiation or mitochondrial biogenesis of periosteum-derived MSCs (PO-MSCs), which play key roles in bone tissue maintenance and fracture healing. Thus, in order to test a possible preventive or therapeutic effect of resveratrol on osteoporosis, this study investigated the effects of resveratrol treatments on osteogenic differentiation and mitochondrial biogenesis of PO-MSCs. METHODS: The optimal doses of resveratrol treatment on PO-MSCs were determined by cell proliferation and viability assays. Osteogenic differentiation of PO-MSCs under resveratrol treatment was assessed by alkaline phosphatase activities (ALP, an early biomarker of osteogenesis) as well as by extracellular calcium deposit levels (a late biomarker). Mitochondrial biogenesis during osteogenic differentiation of PO-MSCs was measured by quantifying both mitochondrial mass and mitochondrial DNA (mtDNA) contents. RESULTS: Resveratrol treatments above 10 µM seem to have negative effects on cell proliferation and viability of PO-MSCs. Resveratrol treatment (at 5 µM) on PO-MSCs during osteogenic differentiation increased both ALP activities and calcium deposits compared to untreated control groups, demonstrating an enhancing effect of resveratrol on osteogenesis. In addition, resveratrol treatment (at 5 µM) during osteogenic differentiation of PO-MSCs increased both mitochondrial mass and mtDNA copy numbers, indicating that resveratrol can bolster mitochondrial biogenesis in the process of PO-MSC osteogenic differentiation. CONCLUSION: Taken together, the findings of this study describe the roles of resveratrol in promoting osteogenesis and mitochondrial biogenesis of human PO-MSCs suggesting a possible application of resveratrol as a supplement for osteoporosis and/or osteoporotic fractures.

Skeletal stem cells: insights into maintaining and regenerating the skeleton.

Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance and repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with inconsistent characteristics. However, recent in vivo tracking experiments have revealed the presence of SSCs not only within the bone marrow but also within the periosteum and growth plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to lineage potential. It has also been revealed that, during digit tip regeneration and in some non-mammalian vertebrates, the dedifferentiation of osteoblasts may contribute to skeletal regeneration. Here, we examine how these research findings have furthered our understanding of the diversity and plasticity of SSCs that mediate skeletal maintenance and repair.

Response of human periosteal cells to degradation products of zinc and its alloy.

Zinc (Zn) and its alloys are proposed as promising resorbable materials for osteosynthesis implants. Detailed studies should be undertaken to clarify their properties in terms of degradability, biocompatibility and osteoinductivity. Degradation products of Zn alloys might affect directly adjacent cellular and tissue responses. Periosteal stem cells are responsible for participating in intramembranous ossification during fracture healing. The present study aims at examining possible effects emanating from Zn or Zn-4Ag (wt%) alloy degradation products on cell viability and osteogenic differentiation of a human immortalized cranial periosteal cell line (TAg cells). Therefore, a modified extraction method was used to investigate the degradation behavior of Zn and Zn-4Ag alloys under cell culture conditions. Compared with pure Zn, Zn-4Ag alloy showed almost fourfold higher degradation rates under cell culture conditions, while the associated degradation products had no adverse effects on cell viability. Osteogenic induction of TAg cells revealed that high concentration extracts significantly reduced calcium deposition of TAg cells, while low concentration extracts enhanced calcium deposition, indicating a dose-dependent effect of Zn ions. Our results give evidence that the observed cytotoxicity effects were determined by the released degradation products of Zn and Zn-4Ag alloys, rather than by degradation rates calculated by weight loss. Extracellular Zn ion concentration was found to modulate osteogenic differentiation of TAg cells. These findings provide significant implications and guidance for the development of Zn-based alloys with an optimized degradation behavior for Zn-based osteosynthesis implants.

Quantitative proteomics analysis of deer antlerogenic periosteal cells reveals potential bioactive factors in velvet antlers.

Velvet antlers (VA) have been used as medicines and nutraceuticals for over 2000 years. Meanwhile, deer antlers are the only mammalian organs that can fully regenerate after annual shedding. The antler formation and regeneration rely on the stem cells resident in antlerogenic periosteum (AP), transplantation of which can induce ectopic antler formation. Here, a comprehensive quantitative proteomic analysis of antlerogenic periosteal cells (AP cells), compared with the adjacent facial periosteal cells (FP cells), was carried out, from both extracellular and intracellular perspectives. In this study, the stable isotope labeling by amino acids in cell culture (SILAC) was applied to ensure the precision of quantification. Then, the protein equalization strategy and reverse-phase liquid chromatography (RPLC) separation in high pH were utilized to improve the depth of proteome profiling. Proteomics analysis of the conditioned media (CM) from AP and FP cells showed that significantly over-expressed extracellular proteins in AP cells were involved in cell proliferation, angiogenesis and neurogenesis. Combining the extracellular and intracellular proteomes, we found several potential secreted proteins might regulate antler formation and regeneration, such as SFRP4 and LUM. These results provide new insight into the underlying mechanism of antler formation and regeneration.

Inhibition of the Prostaglandin EP-1 Receptor in Periosteum Progenitor Cells Enhances Osteoblast Differentiation and Fracture Repair.

Fracture healing is a complex and integrated process that involves mesenchymal progenitor cell (MPC) recruitment, proliferation and differentiation that eventually results in bone regeneration. Prostaglandin E2 (PGE2) is an important regulator of bone metabolism and has an anabolic effect on fracture healing. Prior work from our laboratory showed EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation. We also showed that bone marrow MSCs from EP1-/- mice exhibit increased osteoblastic differentiation in vitro. In this study we investigate the changes in the periosteal derived MPCs (PDMPCs), which are crucial for fracture repair, upon EP1 deletion. EP1-/- PDMPCs exhibit increased numbers of total (CFU-F) and osteoblastic colonies (CFU-O) as well as enhanced osteoblastic and chondrogenic differentiation. Moreover, we tested the possible therapeutic application of a specific EP1 receptor antagonist to accelerate fracture repair. Our findings showed that EP1 antagonist administration to wild type mice in the early stages of repair similarly resulted in enhanced CFU-F, CFU-O, and osteoblast differentiation in PDMPCs and resulted in enhanced fracture callus formation at 10 days post fracture and increased bone volume and improved biomechanical healing of femur fractures at 21 days post fracture.

Real-time detection of antibiotics cytotoxicity in rabbit periosteal cells using microfluidic devices with comparison to conventional culture assays.

BACKGROUND: Local antibiotic application has been widely used in orthopedic surgery. The dose-related toxicity of antibiotics towards periosteal tissues and resulting effects on osteogenic expression are yet to be studied. METHODS: Periosteal cells harvested from the medial tibia of New Zealand White rabbits were used. A seeding density of 5 × 103 cells/cm2 was determined to be optimal for testing in the pilot study; the cells were cultured in xCELLigence 96-well plates. Microfluidic impedance analyzers were used to monitor cellular proliferation in microfluidic culture systems with exposure to three different concentrations (10 µg/mL, 100 µg/mL, and 1000 µg/mL) of cefazolin, ciprofloxacin, and vancomycin, respectively. The correlation of cell index at day 7 with optical density values from WST-1 assays using conventional cultures was evaluated by calculating the Pearson's coefficient. RNA analysis was performed to investigate the expression of osteogenic markers in the cultured cells, including core-binding factor alpha 1 (Cbfa1), osteopontin (OPN), and osteopontin promoter (OPNp), relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control. RESULTS: A significant dose-related inhibition of cell index was found for all the 3 antibiotics, whereas the WST-1 assays showed a significant dose-related inhibition of cellular proliferation only at a high dose of cefazolin (1000 µg/mL) and medium-to-high dose of ciprofloxacin (100 µg/mL and 1000 µg/mL). Pearson's coefficient analysis indicated a high correlation between the cell index and optical density values of WST-1 assays only for medium and high doses of ciprofloxacin (100 µg/mL and 1000 µg/mL); a moderate correlation was seen for cefazolin, and a low dose of ciprofloxacin (10 µg/mL). RNA analysis confirmed significant dose-related inhibition of cfba1, OPN, and OPNp expression by all three antibiotics. CONCLUSION: With optimal seeding amounts, rabbit periosteal cells can be dynamically monitored in the xCELLigence microfluidic system. Dose-related inhibition of cellular proliferation and osteogenic expression was found after exposure to cefazolin and ciprofloxacin. By providing real-time detection and exhibiting comparable correlation, microfluidic impedance-based analyzer is a feasible alternative to the conventional WST-1 assays.

Human Platelet Lysate Improves Bone Forming Potential of Human Progenitor Cells Expanded in Microcarrier-Based Dynamic Culture.

Xenogeneic-free media are required for translating advanced therapeutic medicinal products to the clinics. In addition, process efficiency is crucial for ensuring cost efficiency, especially when considering large-scale production of mesenchymal stem cells (MSCs). Human platelet lysate (HPL) has been increasingly adopted as an alternative for fetal bovine serum (FBS) for MSCs. However, its therapeutic and regenerative potential in vivo is largely unexplored. Herein, we compare the effects of FBS and HPL supplementation for a scalable, microcarrier-based dynamic expansion of human periosteum-derived cells (hPDCs) while assessing their bone forming capacity by subcutaneous implantation in small animal model. We observed that HPL resulted in faster cell proliferation with a total fold increase of 5.2 ± 0.61 in comparison to 2.7 ± 02.22-fold in FBS. Cell viability and trilineage differentiation capability were maintained by HPL, although a suppression of adipogenic differentiation potential was observed. Differences in mRNA expression profiles were also observed between the two on several markers. When implanted, we observed a significant difference between the bone forming capacity of cells expanded in FBS and HPL, with HPL supplementation resulting in almost three times more mineralized tissue within calcium phosphate scaffolds. FBS-expanded cells resulted in a fibrous tissue structure, whereas HPL resulted in mineralized tissue formation, which can be classified as newly formed bone, verified by µCT and histological analysis. We also observed the presence of blood vessels in our explants. In conclusion, we suggest that replacing FBS with HPL in bioreactor-based expansion of hPDCs is an optimal solution that increases expansion efficiency along with promoting bone forming capacity of these cells. Stem Cells Translational Medicine 2019;8:810&821.