Differential Responses to Aging Among the Transcriptome and Proteome of Mesenchymal Progenitor Populations.

The biological aging of stem cells (exhaustion) is proposed to contribute to the development of a variety of age-related conditions. Despite this, little is understood about the specific mechanisms which drive this process. In this study, we assess the transcriptomic and proteomic changes in 3 different populations of mesenchymal progenitor cells from older (50-70 years) and younger (20-40 years) individuals to uncover potential mechanisms driving stem cell exhaustion in mesenchymal tissues. To do this, we harvested primary bone marrow mesenchymal stem and progenitor cells (MPCs), circulating osteoprogenitors (COP), and adipose-derived stem cells (ADSCs) from younger and older donors, with an equal number of samples from men and women. These samples underwent RNA sequencing and label-free proteomic analysis, comparing the younger samples to the older ones. There was a distinct transcriptomic phenotype in the analysis of pooled older stem cells, suggestive of suppressed proliferation and differentiation; however, these changes were not reflected in the proteome of the cells. Analyzed independently, older MPCs had a distinct phenotype in both the transcriptome and proteome consistent with altered differentiation and proliferation with a proinflammatory immune shift in older adults. COP cells showed a transcriptomic shift to proinflammatory signaling but no consistent proteomic phenotype. Similarly, ADSCs displayed transcriptomic shifts in physiologies associated with cell migration, adherence, and immune activation but no proteomic change with age. These results show that there are underlying transcriptomic changes with stem cell aging that may contribute to a decline in tissue regeneration. However, the proteome of the cells was inconsistently regulated.

Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed "Cre-only" Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

The complement factor H-related protein-5 (CFHR5) exacerbates pathological bone formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease, characterized by excessive new bone formation. We previously reported that the complement factor H-related protein-5 (CFHR5), a member of the human factor H protein family, is significantly elevated in patients with AS compared to other rheumatic diseases. However, the pathophysiological mechanism underlying new bone formation by CFHR5 is not fully understood. In this study, we revealed that CFHR5 and proinflammatory cytokines (TNF, IL-6, IL-17A, and IL-23) were elevated in the AS group compared to the HC group. Correlation analysis revealed that CFHR5 levels were not significantly associated with proinflammatory cytokines, while CFHR5 levels in AS were only positively correlated with the high CRP group. Notably, treatment with soluble CFHR5 has no effect on clinical arthritis scores and thickness at hind paw in curdlan-injected SKG, but significantly increased the ectopic bone formation at the calcaneus and tibia bones of the ankle as revealed by micro-CT image and quantification. Basal CFHR5 expression was upregulated in AS-osteoprogenitors compared to control cells. Also, treatment with CFHR5 remarkedly induced bone mineralization status of AS-osteoprogenitors during osteogenic differentiation accompanied by MMP13 expression. We provide the first evidence demonstrating that CFHR5 can exacerbate the pathological bone formation of AS. Therapeutic modulation of CFHR5 could be promising for future treatment of AS. KEY MESSAGES: Serum level of CFHR5 is elevated and positively correlated with high CRP group of AS patients. Recombinant CFHR5 protein contributes to pathological bone formation in in vivo model of AS. CFHR5 is highly expressed in AS-osteoprogenitors compared to disease control. Recombinant CFHR5 protein increased bone mineralization accompanied by MMP13 in vitro model of AS.

Elevated BMPR2 expression amplifies osteoblast differentiation in ankylosing spondylitis.

Objective: Bone morphogenetic protein receptor type 2 (BMPR2) has been associated with radiographic changes in ankylosing spondylitis (AS), but further characterization of the cellular signaling pathway in osteoprogenitor (OP) is not clearly understood. The aim of this study was to investigate the expression of BMPR2 and bone morphogenetic protein 2 (BMP2)-mediated responsibility in AS. Methods: We collected 10 healthy control (HC) and 14 AS-OPs derived from facet joints. Subsequently, we then conducted RNA sequencing with two samples per group and selected BMP-related genes. Facet joint tissues and derived primary OPs were evaluated by validation of selected RNA sequencing data, immunohistochemistry, and comparison of osteogenic differentiation potential. Results: Based on RNA-sequencing analysis, we found that BMPR2 expression is higher in AS-OPs compared to in HC-OPs. We also validated the increased BMPR2 expression in facet joint tissues with AS and its derived OPs in messenger RNA and protein levels. Additionally, primary AS-OPs showed much greater response to osteogenic differentiation induced by BMP2 and a higher capacity for smad1/5/8-induced RUNX2 expression compared to HCs. Conclusion: The expression of BMPR2 was found to be significantly increased in facet joint tissues of patients with AS. These findings suggest that BMPR2 may play a role in the BMP2-mediated progression of AS.

Osteoprogenitor recruitment and differentiation during intracortical bone remodeling of adolescent humans.

BACKGROUND: Recruitment and proliferation of osteoprogenitors during the reversal-resorption phase, and their differentiation into mature bone-forming osteoblasts is crucial for initiation of bone formation during bone remodeling. This study investigates the osteoprogenitors' gradual recruitment, proliferation, and differentiation into bone-forming osteoblasts within intracortical remodeling events of healthy adolescent humans. METHODS: The study was conducted on cortical bone specimens from 11 adolescent human controls - patients undergoing surgery due to coxa valga. The osteoprogenitor recruitment route and differentiation into osteoblasts were backtracked using immunostainings and in situ hybridizations with osteoblastic markers (CD271/NGFR, osterix/SP7, COL3A1 and COL1A1). The osteoblastic cell populations were defined based on the pore surfaces, and their proliferation index (Ki67), density and number/circumference were estimated in multiplex-immunofluorescence (Ki67, TRAcP, CD34) stained sections. RESULTS: During the reversal-resorption phase, osteoclasts are intermixed with (COL3A1+NFGR+) osteoblastic reversal cells, which are considered to be osteoprogenitors of (COL1A1+SP7+) bone-forming osteoblasts. Initiation of bone formation requires a critical density of these osteoprogenitors (43 ± 9 cells/mm), which is reached though proliferation (4.4 ± 0.5 % proliferative) and even more so through recruitment of osteoprogenitors, but challenged by the ongoing expansion of the canal circumference. These osteoprogenitors most likely originate from osteoblastic bone lining cells and mainly lumen osteoprogenitors, which expand their population though proliferation (4.6 ± 0.3 %) and vascular recruitment. These lumen osteoprogenitors resemble canopy cells above trabecular remodeling sites, and like canopy cells they extend above bone-forming osteoblasts where they may rejuvenate the osteoblast population during bone formation. CONCLUSION: Initiation of bone formation during intracortical remodeling requires a critical density of osteoprogenitors on eroded surfaces, which is reached though proliferation and recruitment of local osteoprogenitors: bone lining cells and lumen osteoprogenitors.

AcanCreER lacks specificity to chondrocytes and targets periosteal progenitors in the fractured callus.

Aggrecan (Acan) is a large proteoglycan molecule constituting the extracellular matrix of cartilage, secreted by chondrocytes. To specifically target the chondrocyte lineage, researchers have widely used the AcanCreER mouse model. Evaluation of specificity and efficiency of recombination, requires Cre animals to be crossed with reporter mice. In order to accurately interpret data from Cre models, it is imperative to consider A) the amount of recombination occurring in cells/tissues that are not intended for targeting (i.e., non-specific expression), B) the efficiency of Cre recombination, which can depend on dose and duration of tamoxifen treatment, and C) the activation of CreER without tamoxifen induction, known as "Cre leakage." Using a highly sensitive reporter mouse (Ai9, tdTomato), we performed a comprehensive analysis of the AcanCreER system. Surprisingly, we observed expression in cells within the periosteum. These cells expand at a stage when chondrocytes are not yet present within the forming callus tissue (Acan/Ai9+ cells). In pulse-chase experiments, we confirmed that fibroblastic Acan/Ai9+ cells within the periosteum can directly give rise to osteoblasts. Our results show that Acan/Ai9+ is not specific for the chondrocyte lineage in the fracture callus or with the tibial holes. The expression of AcanCreER in periosteal progenitor cells complicates the interpretation of studies evaluating the transition of chondrocytes to osteoblasts (termed transdifferentiation). Awareness of these issues and the limitations of the system will lead to better data interpretation.

Differential responses to aging amongst the transcriptome and proteome of mesenchymal progenitor populations.

The biological aging of mesenchymal stem cells is proposed to contribute to the development of a range of musculoskeletal and systemic diseases associated with older adults, such as osteoporosis, sarcopenia, and frailty. Despite this, little is understood about the specific mechanisms which drive this stem cell exhaustion, with most studies evaluating indirect effects of other aging changes, such as DNA damage, senescence, and inflammaging. In this study, we assess the transcriptomic and proteomic changes in three different populations of mesenchymal progenitor cells from older (50-70 years) and younger (20-40 years) individuals to uncover potential mechanisms driving stem cell exhaustion in mesenchymal tissues. To do this, we harvested primary bone marrow mesenchymal stem and progenitor cells (MPCs), circulating osteoprogenitors (COP), and adipose-derived stem cells (ADSCs) from younger and older donors, with an equal number of samples from males and females. These samples underwent RNA sequencing and label-free proteomic analysis, comparing the younger samples to the older ones. There was a distinct transcriptomic phenotype associated with the pooled older stem cells, indicative of suppressed proliferation and differentiation; however, there was no consistent change in the proteome of the cells. Older MPCs had a distinct phenotype in both the transcriptome and proteome, again consistent with altered differentiation and proliferation, but also a pro-inflammatory immune shift in older adults. COP cells showed a strong transcriptomic shift to pro-inflammatory signaling but no consistent proteomic phenotype. Similarly, ADSCs displayed transcriptomic shift in physiologies associated with cell migration, adherence, and immune activation, but no consistent proteomic change with age. These results show that there are underlying transcriptomic changes with stem cell aging that likely contribute to a decline in tissue regeneration; however, contextual factors such as the microenvironment and general health status also have a strong role in this.

Reciprocal Alterations in Osteoprogenitor and Immune Cell Populations in Rheumatoid Synovia.

Rheumatoid arthritis (RA) is chronic, autoimmune joint inflammation characterized by irreversible joint destruction. Besides increased resorption, destruction is a result of decreased bone formation, due to suppressed differentiation and function of the mesenchymal lineage-derived osteoblasts in inflammatory milieu. In this study, we analyzed the cellular composition of synovial tissue from 11 RA and 10 control patients harvested during planned surgeries in order to characterize resident synovial progenitor populations. Synovial cells were released by collagenase, and labeled for flow cytometry by two antibody panels: 1. CD3-FITC, CD14-PE, 7-AAD, CD11b-PECy7, CD235a-APC, CD19-APCeF780; and 2. 7-AAD, CD105-PECy7, CD45/CD31/CD235a-APC, and CD200-APCeF780. The proportions of lymphocytes (CD3+, CD19+) and myeloid (CD11b+, CD14+) cells were higher in synovial tissue from the patients with RA than in the controls. Among non-hematopoietic (CD45-CD31-CD235a-) cells, there was a decrease in the proportion of CD200+CD105- and increase in the proportion of CD200-CD105+ cells in synovial tissue from the patients with RA in comparison to the control patients. The proportions of both populations were associated with inflammatory activity and could discriminate between the RA and the controls.

Controlled mechanical loading improves bone regeneration by regulating type H vessels in a S1Pr1-dependent manner.

Despite the best treatment, approximately 10% of fractures still face undesirable repair and result in delayed unions or non-unions. Dynamic mechanical stimulation promotes bone formation, when applied at the correct time frame, with optimal loading magnitude, frequency, and repetition. Controlled mechanical loading significantly increases osteogenic cells during the matrix deposition phase of bone repair. In the bone defect, the blood vessel network guides the initial bone formation activities. A unique blood vessel subtype (Type H) exists in bone, which expresses high levels of CD31 and endomucin, and functions to couple angiogenesis and osteogenesis. However, how this form of controlled mechanical loading regulates the Type H vessels and promotes bone formation is still not clear. Sphingosine 1-phosphate (S1P) participates in the bone anabolic process and is a key regulator of the blood vessel. Its receptor, sphingosine 1-phosphate receptor 1 (S1Pr1), is a mechanosensitive protein that regulates vascular integrity. Therefore, we hypothesis that controlled anabolic mechanical loading promotes bone repair by acting on Type H vessels. To study the effect of S1Pr1 on loading induced-bone repair, we utilized a stabilized tibial defect model, which allows for the application of anabolic mechanical loading. Mechanical loading upregulated S1Pr1 within the entire defect, with up to 80% expressed in blood vessels, as observed by deep tissue imaging. Additionally, S1Pr1 antagonism by W146 inhibited the anabolic effects of mechanical loading. We showed that mechanical loading or activating S1Pr1 could induce YAP nuclear translocation, a key regulator in the cell's mechanical response, in endothelial cells (ECs) in vitro. Inhibition of S1Pr1 in endothelial cells by siRNA reduced loading-induced YAP nuclear translocation and expressions of angiogenic genes. In vivo, YAP nuclear translocation in Type H vessels was up-regulated after mechanical loading but was inhibited by antagonizing S1Pr1. S1Pr1 agonist, FTY720, increased bone volume and Type H vessel volume, similar to that of mechanical stimulation. In conclusion, controlled anabolic mechanical loading enhanced bone formation mainly through Type H vessels in a S1Pr1-dependent manner.

Effect of Alumina Particles on the Osteogenic Ability of Osteoblasts.

Biomaterials are used as implants for bone and dental disabilities. However, wear particles from the implants cause osteolysis following total joint arthroplasty (TJA). Ceramic implants are considered safe and elicit a minimal response to cause periprosthetic osteolysis. However, few reports have highlighted the adverse effect of ceramic particles such as alumina (Al2O3) on various cell types. Hence, we aimed to investigate the effect of Al2O3 particles on osteoprogenitors. A comparative treatment of Al2O3, Ti, and UHMWPE particles to osteoprogenitors at a similar concentration of 200 µg/mL showed that only Al2O3 particles were able to suppress the early and late differentiation markers of osteoprogenitors, including collagen synthesis, alkaline phosphatase (ALP) activity and mRNA expression of Runx2, OSX, Col1α, and OCN. Al2O3 particles even induced inflammation and activated the NFkB signaling pathway in osteoprogenitors. Moreover, bone-forming signals such as the WNT/ß-catenin signaling pathway were inhibited by the Al2O3 particles. Al2O3 particles were found to induce the mRNA expression of WNT/ß-catenin signaling antagonists such as DKK2, WIF, and sFRP1 several times in osteoprogenitors. Taken together, this study highlights a mechanistic view of the effect of Al2O3 particles on osteoprogenitors and suggests therapeutic targets such as NFĸB and WNT signaling pathways for ceramic particle-induced osteolysis.

Exosomal miRNAs from Prostate Cancer Impair Osteoblast Function in Mice.

Prostate cancer (PCa) is the most frequent malignancy in older men with a high propensity for bone metastases. Characteristically, PCa causes osteosclerotic lesions as a result of disrupted bone remodeling. Extracellular vesicles (EVs) participate in PCa progression by conditioning the pre-metastatic niche. However, how EVs mediate the cross-talk between PCa cells and osteoprogenitors in the bone microenvironment remains poorly understood. We found that EVs derived from murine PCa cell line RM1-BM increased metabolic activity, vitality, and cell proliferation of osteoblast precursors by >60%, while significantly impairing mineral deposition (-37%). The latter was further confirmed in two complementary in vivo models of ossification. Accordingly, gene and protein set enrichments of osteoprogenitors exposed to EVs displayed significant downregulation of osteogenic markers and upregulation of proinflammatory factors. Additionally, transcriptomic profiling of PCa-EVs revealed the abundance of three microRNAs, miR-26a-5p, miR-27a-3p, and miR-30e-5p involved in the suppression of BMP-2-induced osteogenesis in vivo, suggesting the critical role of these EV-derived miRNAs in PCa-mediated suppression of osteoblast activity. Taken together, our results indicate the importance of EV cargo in cancer-bone cross-talk in vitro and in vivo and suggest that exosomal miRNAs may contribute to the onset of osteosclerotic bone lesions in PCa.

Development and validation of a new method to isolate, expand, and differentiate circulating osteogenic precursor (COP) cells.

Circulating osteogenic precursor (COP) cells are a population of progenitor cells in the peripheral blood with the capacity to form bone in vitro and in vivo. They have characteristics of the mesenchymal stem and progenitor pool found in the bone marrow; however, more recently, a population of COP cells has been identified with markers of the hematopoietic lineage such as CD45 and CD34. While this population has been associated with several bone pathologies, a lack of cell culture models and inconsistent characterization has limited mechanistic research into their behavior and physiology. In this study, we describe a method for the isolation of CD45+/CD34+/alkaline phosphatase (ALP) + COP cells via fluorescence-activated cell sorting, as well as their expansion and differentiation in culture. Hematopoietic COP cells are a discreet population within the monocyte fraction of the peripheral blood mononuclear cells, which form proliferative, fibroblastoid colonies in culture. Their expression of hematopoietic markers decreases with time in culture, but they express markers of osteogenesis and deposit calcium with differentiation. It is hoped that this will provide a standard for their isolation, for consistency in future research efforts, to allow for the translation of COP cells into clinical settings.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

Teriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

Isolation and establishment of skin-derived and mesenchymal cells from south American bat Artibeus planirostris (Chiroptera - Phyllostomidae).

Animal models represent a crucial tool for biological research, so the establishment of new cultures is fundamental for the discovery of new therapies and the understanding of mechanisms of cell development in the most diverse animals. Here, we report the successful establishment of two new primary cell cultures derived from a South American bat (Artibeus planirostris). The establishment of a new bat culture can help in the investigation of new zoonoses since bats have been proposed as carriers of these diseases. We evaluated the chromosomal stability of cells from different passages. Primary cultures were collected from ear tissues and bone marrow of A. planirostris. Cultures were expanded, and osteogenic and adipogenic inductions were conducted for 21 days. For osteogenic differentiation, the medium was supplemented with 0.1 µM dexamethasone, 3 mM ß-glycerophosphate, and 10 µM L-ascorbic acid 2-phosphate. For adipogenic differentiation, the medium was supplemented with 5 µM rosiglitazone, 0.4 µM insulin, 0.1 mM indomethacin, and 0.1 µM dexamethasone. After the induction period, the cells were stained with Alizarin Red to assess osteogenic differentiation and Oil Red O to assess adipogenic differentiation. We observed the appearance of lipid droplets in adipocytes and the extracellular deposition of calcium matrix by osteocytes, indicating that bone marrow-derived cells and skin-derived cells of A. planirostris could successfully differentiate into these lineages. Also, the number of chromosomes remained stable for both primary cultures during passages 2, 4, 6, and 8.

Increased NF-kB activity in osteoprogenitor-lineage cells impairs the balance of bone versus fat in the marrow of skeletally mature mice.

"Senile osteoporosis" is defined as significant aging-associated bone loss, and is accompanied by increased fat in the bone marrow. The proportion of adipocytes in bone marrow is inversely correlated with bone formation, and is associated with increased risk of fracture. NF-κB is a transcription factor that functions as a master regulator of inflammation and bone remodeling. NF-κB activity increases during aging; furthermore, constitutive activation of NF-κB significantly impairs skeletal development in neonatal mice. However, the effects of NF-κB activation using a skeletally mature animal model have not been examined. In the current study, an osteoprogenitor (OP)-specific, doxycycline-regulated NF-κB activated transgenic mouse model (iNF-κB/OP) was generated to investigate the role of NF-κB in bone remodeling in skeletally mature mice. Reduced osteogenesis in the OP-lineage cells isolated from iNF-κB/OP mice was only observed in the absence of doxycycline in vitro. Bone mineral density in the metaphyseal regions of femurs and tibias was reduced in iNF-κB/OP mice. No significant differences in bone volume fraction and cortical bone thickness were observed. Osmium-stained bone marrow fat was increased in epiphyseal and metaphyseal areas in the tibias of iNF-κB/OP mice. These findings suggest that targeting NF-κB activity as a therapeutic strategy may improve bone healing and prevent aging-associated bone loss in aged patients.

Effects of dihydrotestosterone on osteoblast activity in curdlan-administered SKG mice and osteoprogenitor cells in patients with ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is characteristically male-predominant, and progressive spinal ankylosis affects male patients more severely; however, the hormonal effects in males with AS are poorly understood. METHODS: In the present study, the regulatory effects of dutasteride, a 5-α reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone (DHT), were examined in curdlan-administered male SKG mice to determine spinal bone formation, bone metabolism-related markers, and interleukin (IL)-17A cytokine and T cell populations. In addition, the effects of DHT on primary osteoprogenitors from the facet joints of AS patients were assessed based on osteoblast-related parameters. DHT level was measured, and the correlation with modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) was analyzed in AS patients. RESULTS: In curdlan-administered SKG mice, dutasteride treatment resulted in an increased accumulation of hydroxyapatite in the spine which was positively correlated with serum IL-17A levels. In the analysis of bone metabolism-related molecules, a decrease in sclerostin levels was observed in the sera in the dutasteride group. Continuous exposure to DHT resulted in fewer calcium deposits in AS osteoprogenitors during osteoblast differentiation. DHT-treated AS osteoprogenitors showed decreased osteocalcin and increased DKK1 and SOST1 mRNA expression, supporting the results of the in vivo experiments. Treatment with dutasteride upregulated bone formation in the spine of curdlan-administered SKG mice and DHT treatment downregulated osteoblast differentiation in vitro. CONCLUSIONS: Treatment with dutasteride affected the bone formation in the spine of curdlan-treated SKG mice, and DHT treatment attenuated osteoblast differentiation in vitro. Therefore, contrary to what could be expected if osteoblasts contributed to spinal ankylosis, DHT inhibition might increase rather than decrease the progression of spinal ankylosis despite the higher levels of DHT observed in many AS patients.

Inhibition of the mitochondrial permeability transition improves bone fracture repair.

Bone fracture is accompanied by trauma, mechanical stresses, and inflammation - conditions known to induce the mitochondrial permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening leads to more inflammation, cell death and potentially to disruption of fracture repair. Here we performed a proof-of-concept study and tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. First, our in vitro experiments indicated pro-osteogenic and anti-inflammatory effects in osteoprogenitors upon CypD knock-out or pharmacological inhibition. Using a bone fracture model in mice, we observed that bone formation and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice or wild type mice treated with a CypD inhibitor, NIM811, when compared to controls. These effects were evident in young male but not female mice, however in older (13 month-old) female mice bone formation was also increased during fracture repair. In contrast to global CypD knock-out, mesenchymal lineage-specific (Prx1-Cre driven) CypD deletion did not result in improved fracture repair. Our findings implicate MPTP in bone fracture and suggest systemic CypD inhibition as a modality to promote fracture repair.

Expansion of Bone Precursors through Jun as a Novel Treatment for Osteoporosis-Associated Fractures.

Osteoporosis and osteoporotic fractures lead to decreased life quality and high healthcare costs. Current treatments prevent losses in bone mass and fractures to some extent but have side effects. Therefore, better therapies are needed. This study investigated whether the transcription factor Jun has a specific pro-osteogenic potency and whether modulating Jun could serve as a novel treatment for osteoporosis-associated fractures. We demonstrate that ectopically transplanted whole bones and distinct osteoprogenitors increase bone formation. Perinatal Jun induction disturbs growth plate architecture, causing a striking phenotype with shortened and thickened bones. Molecularly, Jun induces hedgehog signaling in skeletal stem cells. Therapeutically, Jun accelerates bone growth and healing in a drilling-defect model. Altogether, these results demonstrate that Jun drives bone formation by expanding osteoprogenitor populations and forcing them into the bone fate, providing a rationale for future clinical applications.

Nestin-GFP transgene labels skeletal progenitors in the periosteum.

The periosteum is critical for bone repair and contains skeletal stem cells (SSCs), but these cells are still poorly characterized. In the bone marrow, cells expressing the Nes-GFP transgene have been described to be SSCs. Here, we investigated whether Nes-GFP expression also typifies SSCs in the periosteum. We show that in adult mice, Nes-GFP cells are present in the periosteum and localize closely to blood vessels, but periosteal Nes-GFP cells express SSC and progenitor markers differently compared to Nes-GFP cells in the bone marrow. Periosteal Nes-GFP cells show in vitro clonogenicity and tri-lineage differentiation potential and they can form bone in vivo. Shortly after fracture, they start to proliferate and they contribute to the osteoblast pool during the repair process. However, periosteal Nes-GFP cells are not slow dividing nor self-renewing in vivo. These results indicate that in adult mice, periosteal Nes-GFP expressing cells are skeletal progenitors rather than true SSCs, and they participate in the fracture healing process.

Skeletal Stem Cells-A Paradigm Shift in the Field of Craniofacial Bone Tissue Engineering.

Defects of the craniofacial skeleton arise as a direct result of trauma, diseases, oncological resection, or congenital anomalies. Current treatment options are limited, highlighting the importance for developing new strategies to restore form, function, and aesthetics of missing or damaged bone in the face and the cranium. For optimal reconstruction, the goal is to replace "like with like." With the inherent challenges of existing options, there is a clear need to develop alternative strategies to reconstruct the craniofacial skeleton. The success of mesenchymal stem cell-based approaches has been hampered by high heterogeneity of transplanted cell populations with inconsistent preclinical and clinical trial outcomes. Here, we discuss the novel characterization and isolation of mouse skeletal stem cell (SSC) populations and their response to injury, systemic disease, and how their re-activation in vivo can contribute to tissue regeneration. These studies led to the characterization of human SSCs which are able to self-renew, give rise to increasingly fate restricted progenitors, and differentiate into bone, cartilage, and bone marrow stroma, all on the clonal level in vivo without prior in vitro culture. SSCs hold great potential for implementation in craniofacial bone tissue engineering and regenerative medicine. As we begin to better understand the diversity and the nature of skeletal stem and progenitor cells, there is a tangible future whereby a subset of human adult SSCs can be readily purified from bone or activated in situ with broad potential applications in craniofacial tissue engineering.