Targeted Disruption of NF1 in Osteocytes Increases FGF23 and Osteoid With Osteomalacia-like Bone Phenotype.

Neurofibromatosis type 1 (NF1, OMIM 162200), caused by NF1 gene mutations, exhibits multi-system abnormalities, including skeletal deformities in humans. Osteocytes play critical roles in controlling bone modeling and remodeling. However, the role of neurofibromin, the protein product of the NF1 gene, in osteocytes is largely unknown. This study investigated the role of neurofibromin in osteocytes by disrupting Nf1 under the Dmp1-promoter. The conditional knockout (Nf1 cKO) mice displayed serum profile of a metabolic bone disorder with an osteomalacia-like bone phenotype. Serum FGF23 levels were 4 times increased in cKO mice compared with age-matched controls. In addition, calcium-phosphorus metabolism was significantly altered (calcium reduced; phosphorus reduced; parathyroid hormone [PTH] increased; 1,25(OH)2 D decreased). Bone histomorphometry showed dramatically increased osteoid parameters, including osteoid volume, surface, and thickness. Dynamic bone histomorphometry revealed reduced bone formation rate and mineral apposition rate in the cKO mice. TRAP staining showed a reduced osteoclast number. Micro-CT demonstrated thinner and porous cortical bones in the cKO mice, in which osteocyte dendrites were disorganized as assessed by electron microscopy. Interestingly, the cKO mice exhibited spontaneous fractures in long bones, as found in NF1 patients. Mechanical testing of femora revealed significantly reduced maximum force and stiffness. Immunohistochemistry showed significantly increased FGF23 protein in the cKO bones. Moreover, primary osteocytes from cKO femora showed about eightfold increase in FGF23 mRNA levels compared with control cells. The upregulation of FGF23 was specifically and significantly inhibited by PI3K inhibitor Ly294002, indicating upregulation of FGF23 through PI3K in Nf1-deficient osteocytes. Taken together, these results indicate that Nf1 deficiency in osteocytes dramatically increases FGF23 production and causes a mineralization defect (ie, hyperosteoidosis) via the alteration of calcium-phosphorus metabolism. This study demonstrates critical roles of neurofibromin in osteocytes for osteoid mineralization. © 2017 American Society for Bone and Mineral Research.

Necrotic Bone Stimulates Proinflammatory Responses in Macrophages through the Activation of Toll-Like Receptor 4.

In Legg-Calvé-Perthes disease, loss of blood supply results in ischemic osteonecrosis of the femoral head (ONFH). Generally, macrophages play important roles in inflammatory responses to tissue necrosis, but their role in ONFH is not known. The purpose of this study was to determine the macrophage-inflammatory responses after ONFH and the receptor mechanisms involved in sensing the necrotic bone, using a piglet model of Legg-Calvé-Perthes disease. Induction of ONFH resulted in increased numbers of CD14+ macrophages in the fibrovascular repair tissue compared with normal, as determined by immunohistochemistry. Quantitative real-time PCR analysis of macrophages isolated by laser capture microdissection showed significantly increased expression of proinflammatory cytokines IL-1ß, tumor necrosis factor-α, and IL-6 in ONFH compared with normal. Because Toll-like receptors (TLRs) mediate macrophage-inflammatory responses in other inflammatory conditions, we determined their gene expression in macrophages and found significantly increased levels of TLR4 but not TLR2 and TLR9 in ONFH. Mechanistically, in vitro, bone marrow-derived macrophages treated with necrotic bone showed increased extracellular signal-regulated kinases 1/2 and Iκ kinase-α phosphorylation, increased proliferation, migration, and inflammatory cytokine expression, which were blocked by TLR4 inhibitor, TAK242, and by TLR4 ablation in macrophages using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease method. In conclusion, necrotic bone stimulates macrophage-inflammatory responses through TLR4 activation.

Targeted disruption of BMP signaling through type IA receptor (BMPR1A) in osteocyte suppresses SOST and RANKL, leading to dramatic increase in bone mass, bone mineral density and mechanical strength.

Recent studies suggest a critical role of osteocytes in controlling skeletal development and bone remodeling although the molecular mechanism is largely unknown. This study investigated BMP signaling in osteocytes by disrupting Bmpr1a under the Dmp1-promoter. The conditional knockout (cKO) mice displayed a striking osteosclerotic phenotype with increased trabecular bone volume, thickness, number, and mineral density as assessed by X-ray and micro-CT. The bone histomorphometry, H&E, and TRAP staining revealed a dramatic increase in trabecular and cortical bone masses but a sharp reduction in osteoclast number. Moreover, there was an increase in BrdU positive osteocytes (2-5-fold) and osteoid volume (~4-fold) but a decrease in the bone formation rate (~85%) in the cKO bones, indicating a defective mineralization. The SEM analysis revealed poorly formed osteocytes: a sharp increase in cell numbers, a great reduction in cell dendrites, and a remarkable change in the cell distribution pattern. Molecular studies demonstrated a significant decrease in the Sost mRNA levels in bone (>95%), and the SOST protein levels in serum (~85%) and bone matrices. There was a significant increase in the ß-catenin (>3-fold) mRNA levels as well as its target genes Tcf1 (>6-fold) and Tcf3 (~2-fold) in the cKO bones. We also showed a significant decrease in the RANKL levels of serum proteins (~65%) and bone mRNA (~57%), and a significant increase in the Opg mRNA levels (>20-fold) together with a significant reduction in the Rankl/Opg ratio (>95%), which are responsible for a sharp reduction in the cKO osteoclasts. The values of mechanical strength were higher in cKO femora (i.e. max force, displacement, and work failure). These results suggest that loss of BMP signaling specifically in osteocytes dramatically increases bone mass presumably through simultaneous inhibition of RANKL and SOST, leading to osteoclast inhibition and Wnt activation together. Finally, a working hypothesis is proposed to explain how BMPR1A controls bone remodeling by inhibiting cell proliferation and stimulating differentiation. It is reported that RANKL and SOST are abundantly expressed by osteocytes. Thus, BMP signaling through BMPR1A plays important roles in osteocytes.

Intraosseous Delivery of Bone Morphogenic Protein-2 Using a Self-Assembling Peptide Hydrogel.

Osteonecrosis of the femoral head (ONFH) is a debilitating hip disorder, which often produces a permanent femoral head deformity and osteoarthritis. The local delivery of biological agents capable of stimulating bone healing offer potential new treatment options for patients with ONFH. Previous studies from our laboratory have shown that a local intraosseous infusion of bone morphogenic protein-2 (BMP-2) was effective in stimulating new bone formation in a piglet model of ischemic ONFH. However, infusion of BMP-2 solution was associated with unwanted dissemination of BMP-2 out of the femoral head and produced heterotopic ossification in the hip capsule. Injectable hydrogels offer a potential method to control the dissemination of biological molecules in vivo. In the present study, we evaluated the potential of a peptide-based, self-assembling hydrogel called RADA16 to transition from a solution to a gel following infusion into the femoral head, thereby preventing backflow, as well as its potential use as a delivery vehicle for BMP-2. Cadaver pig femoral heads were used to study the backflow and the distribution of RADA16 following an intraosseous infusion. Microcomputed tomography analysis following the infusion of RADA16 mixed with a radiocontrast agent revealed a significant decrease in the amount of back flow of radiocontrast agent down the needle track compared to the soluble infusion of radiocontrast without RADA16. Furthermore, RADA16 mixed with radiocontrast agent showed good distribution within the femoral head. In addition, in vitro experiments revealed that higher concentrations of RADA16 decreased the rate of BMP-2 dissemination out of the hydrogel. The BMP-2 that was released from RADA16 maintains its biological activity, inducing the phosphorylation of SMAD1/5/8 in pig primary bone marrow stromal cells. Lastly, pig primary bone marrow stromal cells showed significantly increased in vitro proliferation on RADA16 hydrogels over 1 week compared to tissue culture plastic, suggesting that it is a suitable matrix for supporting cellular proliferation. In conclusion, RADA16 showed potential for use as a drug delivery vehicle to control the delivery of BMP-2 within the femoral head. This novel therapy may be able to provide benefits to patients suffering from debilitating conditions such as osteonecrosis of the femoral head.

In vivo monitoring of activated macrophages and neutrophils in response to ischemic osteonecrosis in a mouse model.

Ischemic osteonecrosis (IO) is caused by disruption of the blood supply to bone. It is a debilitating condition with pathological healing characterized by excessive bone resorption and delayed osteogenesis. Although the majority of research has focused on the role of osteoblasts and osteoclasts in the disease progression, we hypothesize that innate immune cells, macrophages and neutrophils, play a significant role. With the recent development of real-time imaging probes for neutrophils and macrophages, the purpose of this study was to investigate the kinetic immune cell response in a mouse model of IO. Our results show that induction of IO leads to a significant accumulation of activated neutrophils and macrophages at the affected tissue by 48 h after surgery. Additionally, the accumulation of these immune cells remained elevated in comparison to sham controls for up to 6 weeks, indicative of chronic inflammation. Immunohistochemistry confirmed the immune cell infiltration into the necrotic bone marrow and the increased presence of TNFα-positive cells, demonstrating, for the first time, a direct response of these cells to ischemia induced necrotic bone. These new findings support a hypothesis that IO is an osteoimmunologic condition where innate immune cells play a significant role in the chronic inflammation.

Delivery of platelet-derived growth factor as a chemotactic factor for mesenchymal stem cells by bone-mimetic electrospun scaffolds.

The recruitment of mesenchymal stem cells (MSCs) is a vital step in the bone healing process, and hence the functionalization of osteogenic biomaterials with chemotactic factors constitutes an important effort in the tissue engineering field. Previously we determined that bone-mimetic electrospun scaffolds composed of polycaprolactone, collagen I and nanohydroxyapatite (PCL/col/HA) supported greater MSC adhesion, proliferation and activation of integrin-related signaling cascades than scaffolds composed of PCL or collagen I alone. In the current study we investigated the capacity of bone-mimetic scaffolds to serve as carriers for delivery of an MSC chemotactic factor. In initial studies, we compared MSC chemotaxis toward a variety of molecules including PDGF-AB, PDGF-BB, BMP2, and a mixture of the chemokines SDF-1α, CXCL16, MIP-1α, MIP-1ß, and RANTES. Transwell migration assays indicated that, of these factors, PDGF-BB was the most effective in stimulating MSC migration. We next evaluated the capacity of PCL/col/HA scaffolds, compared with PCL scaffolds, to adsorb and release PDGF-BB. We found that significantly more PDGF- BB was adsorbed to, and subsequently released from, PCL/col/HA scaffolds, with sustained release extending over an 8-week interval. The PDGF-BB released was chemotactically active in transwell migration assays, indicating that bioactivity was not diminished by adsorption to the biomaterial. Complementing these studies, we developed a new type of migration assay in which the PDGF-BB-coated bone-mimetic substrates were placed 1.5 cm away from the cell migration front. These experiments confirmed the ability of PDGF-BB-coated PCL/col/HA scaffolds to induce significant MSC chemotaxis under more stringent conditions than standard types of migration assays. Our collective results substantiate the efficacy of PDGF-BB in stimulating MSC recruitment, and further show that the incorporation of native bone molecules, collagen I and nanoHA, into electrospun scaffolds not only enhances MSC adhesion and proliferation, but also increases the amount of PDGF-BB that can be delivered from scaffolds.

Increasing the pore sizes of bone-mimetic electrospun scaffolds comprised of polycaprolactone, collagen I and hydroxyapatite to enhance cell infiltration.

Bone-mimetic electrospun scaffolds consisting of polycaprolactone (PCL), collagen I and nanoparticulate hydroxyapatite (HA) have previously been shown to support the adhesion, integrin-related signaling and proliferation of mesenchymal stem cells (MSCs), suggesting these matrices serve as promising degradable substrates for osteoregeneration. However, the small pore sizes in electrospun scaffolds hinder cell infiltration in vitro and tissue-ingrowth into the scaffold in vivo, limiting their clinical potential. In this study, three separate techniques were evaluated for their capability to increase the pore size of the PCL/col I/nanoHA scaffolds: limited protease digestion, decreasing the fiber packing density during electrospinning, and inclusion of sacrificial fibers of the water-soluble polymer PEO. The PEO sacrificial fiber approach was found to be the most effective in increasing scaffold pore size. Furthermore, the use of sacrificial fibers promoted increased MSC infiltration into the scaffolds, as well as greater infiltration of endogenous cells within bone upon placement of scaffolds within calvarial organ cultures. These collective findings support the use of sacrificial PEO fibers as a means to increase the porosity of complex, bone-mimicking electrospun scaffolds, thereby enhancing tissue regenerative processes that depend upon cell infiltration, such as vascularization and replacement of the scaffold with native bone tissue.

Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite.

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.

Enhancement of peptide coupling to hydroxyapatite and implant osseointegration through collagen mimetic peptide modified with a polyglutamate domain.

Hydroxyapatite (HA) is a widely-used biomaterial for bone repair due to its high degree of osteoconductivity. However, strategies for improving HA performance by functionalizing surfaces with bioactive factors are limited. In this study, we explored the use of a HA-binding domain (heptaglutamate, "E7") to facilitate coupling of the collagen mimetic peptide, DGEA, to two types of HA-containing materials, solid HA disks and electrospun polycaprolactone matrices incorporating nanoparticulate HA. We found that the E7 domain directed significantly more peptide to the surface of HA and enhanced peptide retention on both materials in vitro. Moreover, E7-modified peptides were retained in vivo for at least two months, highlighting the potential of this mechanism as a sustained delivery system for bioactive peptides. Most importantly, E7-DGEA-coupled HA, as compared with DGEA-HA, enhanced the adhesion and osteoblastic differentiation of mesenchymal stem cells, and also increased new bone formation and direct bone-implant contact on HA disks implanted into rat tibiae. Collectively, these results support the use of E7-DGEA peptides to promote osteogenesis on HA substrates, and further suggest that the E7 domain can serve as a universal tool for anchoring a wide variety of bone regenerative molecules to any type of HA-containing material.

The effect of collagen I mimetic peptides on mesenchymal stem cell adhesion and differentiation, and on bone formation at hydroxyapatite surfaces.

Integrin-binding peptides increase cell adhesion to naive hydroxyapatite (HA), however, in the body, HA becomes rapidly modified by protein adsorption. Previously we reported that, when combined with an adsorbed protein layer, RGD peptides interfered with cell adhesion to HA. In the current study we evaluated mesenchymal stem cell (MSC) interactions with HA disks coated with the collagen-mimetic peptides, DGEA, P15 and GFOGER. MSCs adhered equally well to disks coated with DGEA, P15, or collagen I, and all three substrates, but not GFOGER, supported greater cell adhesion than uncoated HA. When peptide-coated disks were overcoated with proteins from serum or the tibial microenvironment, collagen mimetics did not inhibit MSC adhesion, as was observed with RGD, however neither did they enhance adhesion. Given that activation of collagen-selective integrins stimulates osteoblastic differentiation, we monitored osteocalcin secretion and alkaline phosphatase activity from MSCs adherent to DGEA or P15-coated disks. Both of these osteoblastic markers were upregulated by DGEA and P15, in the presence and absence of differentiation-inducing media. Finally, bone formation on HA tibial implants was increased by the collagen mimetics. Collectively these results suggest that collagen-mimetic peptides improve osseointegration of HA, most probably by stimulating osteoblastic differentiation, rather than adhesion, of MSCs.

The effect of RGD peptides on osseointegration of hydroxyapatite biomaterials.

Given that hydroxyapatite (HA) biomaterials are highly efficient at adsorbing proadhesive proteins, we questioned whether functionalizing HA with RGD peptides would have any benefit. In this study, we implanted uncoated or RGD-coated HA disks into rat tibiae for 30 min to allow endogenous protein adsorption, and then evaluated mesenchymal stem cell (MSC) interactions with the retrieved disks. These experiments revealed that RGD, when presented in combination with adsorbed tibial proteins (including fibronectin, vitronectin and fibrinogen), has a markedly detrimental effect on MSC adhesion and survival. Moreover, analyses of HA disks implanted for 5 days showed that RGD significantly inhibits total bone formation as well as the amount of new bone directly contacting the implant perimeter. Thus, RGD, which is widely believed to promote cell/biomaterial interactions, has a negative effect on HA implant performance. Collectively these results suggest that, for biomaterials that are highly interactive with the tissue microenvironment, the ultimate effects of RGD will depend upon how signaling from this peptide integrates with endogenous processes such as protein adsorption.

The tendency of individuals to transfer DNA to handled items.

This research investigates factors influencing the transfer of DNA to handled objects and the process known as 'shedding'. Volunteers were recruited to hold sterile plastic tubes using experiments originally designed by Lowe et al. [A. Lowe, C. Murray, J. Whitaker, G. Tully, P. Gill, The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces, Forensic Sci. Int. 129 (2002) 25-34]. Transferred cellular material was collected from the tubes and STR profiles generated using the AmpFlSTR SGM Plus multiplex with 28 and 34 PCR cycles. Volunteers were asked to hold the tubes with each hand, and to participate in a series of handwashing experiments. The DNA profiling results obtained from the transferred skin cells were compared. An attempt was made to characterize the volunteers as 'good' or 'bad' shedders and to establish which, if any, of the experimental variables were associated with 'good' shedding. Our results suggest that many factors significantly influence shedding, including which hand an individual touches an item with and the time that has elapsed since they last washed their hands. We have found that it may be more complicated than previously reported to categorise a person as being either a 'good' or a 'bad' shedder and that if truly 'good' shedders exist they may be significantly rarer than some have estimated. In the current research no 'good' shedders were observed in a group of 60 volunteers. Given these results, it seems that rather than being applied to individual forensic cases, knowledge of shedding characteristics will be most useful in providing general background data for the interpretation of trace DNA evidence.