During craniofacial development, the Hedgehog (HH) signaling pathway is essential for mesodermal tissue patterning and differentiation. The HH family consists of three protein ligands: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH), of which two are expressed in the craniofacial complex (IHH and SHH). Dysregulations in HH signaling are well documented to result in a wide range of craniofacial abnormalities, including holoprosencephaly (HPE), hypotelorism, and cleft lip/palate. Furthermore, mutations in HH effectors, co-receptors, and ciliary proteins result in skeletal and craniofacial deformities. Cranial suture morphogenesis is a delicate developmental process that requires control of cell commitment, proliferation and differentiation. This review focuses on both what is known and what remains unknown regarding HH signaling in cranial suture morphogenesis and intramembranous ossification. As demonstrated from murine studies, expression of both SHH and IHH is critical to the formation and fusion of the cranial sutures and calvarial ossification. SHH expression has been observed in the cranial suture mesenchyme and its precise function is not fully defined, although some postulate SHH to delay cranial suture fusion. IHH expression is mainly found on the osteogenic fronts of the calvarial bones, and functions to induce cell proliferation and differentiation. Unfortunately, neonatal lethality of IHH deficient mice precludes a detailed examination of their postnatal calvarial phenotype. In summary, a number of basic questions are yet to be answered regarding domains of expression, developmental role, and functional overlap of HH morphogens in the calvaria. Nevertheless, SHH and IHH ligands are integral to cranial suture development and regulation of calvarial ossification. When HH signaling goes awry, the resultant suite of morphologic abnormalities highlights the important roles of HH signaling in cranial development.

An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146 + , CD34 -, and CD45 -), which surround capillaries and microvessels, and (2) adventitial cells (CD146 -, CD34 + , and CD45 -), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 mg/mL), hPSC (2.5 · 10 5 cells), or hPSC + NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC + NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patientmatched samples. Finally, we observed an additive effect on implant vascularization with hPSC + NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC + NELL-1 in the development of strategies for vascularized bone regeneration.

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the proosteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N + NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.

A theoretical inverse relationship has long been postulated for osteogenic and adipogenic differentiation (bone versus adipose tissue differentiation). This inverse relationship in theory at least partially underlies the clinical entity of osteoporosis, in which marrow mesenchymal stem cells (MSCs) have a predilection for adipose differentiation that increases with age. In the present study, we assayed the potential anti-adipogenic effects of Nell-1 protein (an osteoinductive molecule). Using 3T3-L1 (a human preadipocyte cell line) cells and human adipose-derived stromal cells (ASCs), we observed that adenoviral delivered (Ad)-Nell-1 or recombinant NELL-1 protein significantly reduced adipose differentiation across all markers examined (Oil red O staining, adipogenic gene expression [Pparg, Lpl, Ap2]). In a prospective fashion, Hedgehog signaling was assayed as potentially downstream of Nell-1 signaling in regulating osteogenic over adipogenic differentiation. In comparison to Ad-LacZ control, Ad-Nell-1 increased expression of hedgehog signaling markers (Ihh, Gli1, Ptc1). These studies suggest that Nell-1 is a potent anti-adipogenic agent. Moreover, Nell-1 signaling may inhibit adipogenic differentiation via a Hedgehog dependent mechanism.

Summary What is known Cyproheptadine is a serotonin and histamine antagonist that has been suggested as a treatment for serotonin syndrome in case reports. Objective We sought to examine the differences between outcomes and treatment recommendations in patients who received and did not receive cyproheptadine for a probable serotonin syndrome. Methods A retrospective review of cases reported to the California Poison Control System between 2006 and 2017 involving cyproheptadine administration or consideration for treatment of a probable serotonin syndrome. Results and discussion A total of 1420 cases were identified and 288 cases met the inclusion criteria. Of these, 68 (23.1%) patients received cyproheptadine treatment and were significantly older (mean age 49.7 vs 33.5 years, P < 0.00001), intubated (n = 35, 51% vs n = 62, 28%, P < 0.05) and, although not statistically significant, were more frequently admitted to a critical care unit (n = 56, 82.3% vs n = 154, 70.0%, P = 0.09). There were no significant differences in serious outcomes (moderate or worse effects) or hospitalization rates (OR, 1.09, 95% CI, 0.49‐2.64 and OR, 1.99, 95% CI, 0.86‐4.58). There were eight fatalities, of which two patients received cyproheptadine. All fatalities were acute polypharmacy ingestions and had manifested severe symptoms (seizures, hypotension or hyperthermia) either prior to the administration or consideration of cyproheptadine therapy. Cyproheptadine was not administered in 138 (48%) cases primarily due to minimal clinical severity and patient improvement (43%), and not recommended in 82 (28%) cases for reasons from waiting for response to other supportive measures (30%), limited evidence of efficacy (28%) and undetermined diagnosis (14.6%). What is new and conclusion The benefits of and indications for cyproheptadine are uncertain and questionable for the management of a serotonin syndrome. Future recommendations on its use should be based on diagnostic criteria, severity of symptoms and management in conjunction with other supportive measures.

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