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Periodontitis is a prevalent chronic, destructive inflammatory disease affecting tooth-supporting tissues in humans. Guided tissue regeneration strategies are widely utilized for periodontal tissue regeneration generally by using a periodontal membrane. The main role of these membranes is to establish a mechanical barrier that prevents the apical migration of the gingival epithelium and hence allowing the growth of periodontal ligament and bone tissue to selectively repopulate the root surface. Currently available membranes have limited bioactivity and regeneration potential. To address such challenges, an osteoconductive, antibacterial, and flexible poly(caprolactone) (PCL) composite membrane containing zinc oxide (ZnO) nanoparticles is developed. The membranes are fabricated through electrospinning of PCL and ZnO particles. The physical properties, mechanical characteristics, and in vitro degradation of the engineered membrane are studied in detail. Also, the osteoconductivity and antibacterial properties of the developed membrane are analyzed in vitro. Moreover, the functionality of the membrane is evaluated with a rat periodontal defect model. The results confirmed that the engineered membrane exerts both osteoconductive and antibacterial properties, demonstrating its great potential for periodontal tissue engineering.

Objectives: The aim of the ITI Consensus Workshop on zygomatic implants was to provide Consensus Statements and Clinical Recommendations for the use of zygomatic implants., Materials and Methods: Three systematic reviews and one narrative review were written to address focused questions on (1) the indications for the use of zygomatic implants; (2) the survival rates and complications associated with surgery in zygomatic implant placement; (3) long-term survival rates of zygomatic implants and (4) the biomechanical principles involved when zygoma implants are placed under functional loads. Based on the reviews, three working groups then developed Consensus Statements and Clinical Recommendations. These were discussed in a plenary and finalized in Delphi rounds., Results: A total of 21 Consensus Statements were developed from the systematic reviews. Additionally, the group developed 17 Clinical Recommendations based on the Consensus Statements and the combined expertise of the participants., Conclusions: Zygomatic implants are mainly indicated in cases with maxillary bone atrophy or deficiency. Long-term mean zygomatic implant survival was 96.2% [95% CI 93.8; 97.7] over a mean follow-up of 75.4 months (6.3 years) with a follow-up range of 36-141.6 months (3-11.8 years). Immediate loading showed a statistically significant increase in survival over delayed loading. Sinusitis presented with a total prevalence of 14.2% [95% CI 8.8; 22.0] over a mean 65.4 months follow-up, representing the most common complication which may lead to zygomatic implant loss. The international experts suggested clinical recommendations regarding planning, surgery, restoration, outcomes, and the patient's perspective., (© 2023. Deutsche Gesellschaft für Implantologie im Zahn‐, Mund‐ und Kieferbereich e.V., Japanese Society of Oral Implantology.)

Purpose: The purpose of this systematic review was to assess the evidence regarding the indications for placement of zygomatic implants to rehabilitate edentulous maxillae., Material and Methods: A focused question using the PIO format was developed, questioning "in patients in need of an implant-supported rehabilitation of the edentulous maxillae, what are the indications for the use of zygomatic implants''. The primary information analyzed and collected was a clear description of the indication for the use of zygomatic implants., Results: A total of 1266 records were identified through database searching. The full-text review was conducted for 117 papers, and 10 were selected to be included in this review. Zygomatic implant indications were extreme bone atrophy or deficiency secondary to different factors. The quad zygoma concept (two zygomatic implants bilaterally placed and splinted) was applied to 107 patients, the classic zygoma concept (one zygomatic implant bilaterally placed and splinted to standard anterior implants) was used in 88 patients, and the unilateral concept (one zygomatic implant on one side, splinted with one or more conventional implants) was employed in 14 patients., Conclusions: The main indication for the use of zygomatic implants was considered extreme maxillary bone atrophy, resulting from many factors. The clear definition of what was considered "extreme bone atrophy" is not uniquely defined in each paper. Further studies are needed to develop clear indications for zygomatic implants., (© 2023. The Author(s).)

Medication-related osteonecrosis of the jaw (MRONJ) is a severe complication of antiresorptive or antiangiogenic medications, used in the treatment of bone malignancy or osteoporosis. Bone necrosis, mainly represented by osteocytic death, is always present in MRONJ sites; however, the role of osteocyte death in MRONJ pathogenesis is unknown. High mobility group box 1 (HMGB1) is a non-histone nucleoprotein that in its acetylated form accumulates in the cytoplasm, whereas non-acetylated HMGB1 localizes in the nucleus. SIRT1 deacetylase regulates cellular localization of HMGB1. Interestingly, HMGB1 is released during cell necrosis and promotes inflammation through signaling cascades, including activation of the RAGE receptor. Here, we utilized a well-established mouse MRONJ model that utilizes ligature-induced experimental periodontitis (EP) and treatment with either vehicle or zolendronic acid (ZA). Initially, we evaluated HMGB1-SIRT1 expression in osteocytes at 1, 2, and 4 weeks of treatment. Significantly increased cytoplasmic and perilacunar HMGB1 expression was observed at EP sites of ZA versus vehicle (Veh) animals at all time points. SIRT1 colocalized with cytoplasmic HMGB1 and presented a statistically significant increased expression at the EP sites of ZA animals for all time points. RAGE expression was significantly higher in the submucosal tissues EP sites of ZA animals compared with those in vehicle group. To explore the significance of increased cytoplasmic and extracellular HMGB1 and increased RAGE expression in MRONJ pathogenesis, we used pharmacologic inhibitors of these molecules. Combined HMGB1/RAGE inhibition resulted in lower MRONJ incidence with statistically significant decrease in osteonecrotic areas and bone exposure versus non-inhibitor treated ZA animals. Together, our data point to the role of HMGB1 as a central alarmin, overexpressed at early phase of MRONJ pathogenesis during osteocytic death. Moreover, HMGB1-RAGE pathway may represent a new promising therapeutic target in patients at high risk of MRONJ. © 2022 American Society for Bone and Mineral Research (ASBMR)., (© 2022 American Society for Bone and Mineral Research (ASBMR).)

Periodontal diseases are caused by microbial infection and the recruitment of destructive immune cells. Current therapies mainly deal with bacteria elimination, but the regeneration of periodontal tissues remains a challenge. Here we developed a modular microneedle (MN) patch that delivered both antibiotic and cytokines into the local gingival tissue to achieve immunomodulation and tissue regeneration. This MN patch included a quickly dissolvable gelatin membrane for an immediate release of tetracycline and biodegradable GelMA MNs that contained tetracycline-loaded poly(lactic-co-glycolic acid) nanoparticles and cytokine-loaded silica microparticles for a sustained release. Antibiotic release completely inhibited bacteria growth, and the release of IL-4 and TGF-β induced the repolarization of anti-inflammatory macrophages and the formation of regulatory T cells in vitro . In vivo delivery of MN patch into periodontal tissues suppressed proinflammatory factors and promoted pro-regenerative signals and tissue healing, which demonstrated the therapeutic potential of local immunomodulation for tissue regeneration., Competing Interests: Declaration of Interests: S.L., M.M.H.-S., T.A., and A.M. have patent applications (periodontal micropatch and uses thereof, U.S. Provisional Patent Application PCT/US20/58069) related to the current study and, thus, may have related financial interests. The other authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Osteoporosis is a highly prevalent public health burden associated with an increased risk of bone fracture, particularly in aging women. Estrogen, an important medicinal component for the preventative and therapeutic treatment of postmenopausal osteoporosis, induces osteogenesis by activating the estrogen receptor signaling pathway and upregulating the expression of osteogenic genes, such as bone morphogenetic proteins (BMPs). The epigenetic regulation of estrogen-mediated osteogenesis, however, is still unclear. In this report, we found that estrogen significantly induced the expression of lysine-specific demethylase 6B (KDM6B) and that KDM6B depletion by shRNAs led to a significant reduction in the osteogenic potential of DMSCs. Mechanistically, upon estrogen stimulation, estrogen receptor-α (ERα) was recruited to the KDM6B promoter, directly enhancing KDM6B expression. Subsequently, KDM6B was recruited to the BMP2 and HOXC6 promoters, resulting in the removal of H3K27me3 marks and activating the transcription of BMP2 and HOXC6, the master genes of osteogenic differentiation. Furthermore, we found that estrogen enhanced DMSC osteogenesis during calvarial bone regeneration and that estrogen's pro-osteogenic effect was dependent on KDM6B in vivo. Taken together, our results demonstrate the vital role of the ERα/KDM6B regulatory axis in the epigenetic regulation of the estrogen-dependent osteogenic response., (© 2022. The Author(s).)

Biomaterial delivery of bioactive agents and manipulation of stem cell fate are an attractive approach to promote tissue regeneration. Here, smoothened agonist sterosome is developed using small-molecule activators [20 S -hydroxycholesterol (OHC) and purmorphamine (PUR)] of the smoothened protein in the hedgehog pathway as carrier and cargo. Sterosome presents inherent osteoinductive property even without drug loading. Sterosome is covalently immobilized onto three-dimensional scaffolds via a bioinspired polydopamine intermediate to fabricate a hybrid scaffold for bone regeneration. Sterosome-immobilized hybrid scaffold not only provides a favorable substrate for cell adhesion and proliferation but also delivers bioactive agents in a sustained and spatially targeted manner. Furthermore, this scaffold significantly improves osteogenic differentiation of bone marrow stem cells through OHC/PUR-mediated synergistic activation of the hedgehog pathway and also enhances bone repair in a mouse calvarial defect model. This system serves as a versatile biomaterial platform for many applications, including therapeutic delivery and endogenous regenerative medicine., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Injectable hydrogels can fill irregular defects and promote in situ tissue regrowth and regeneration. The ability of directing stem cell differentiation in a three-dimensional microenvironment for bone regeneration remains a challenge. In this study, we successfully nanoengineer an interconnected microporous networked photocrosslinkable chitosan in situ-forming hydrogel by introducing two-dimensional nanoclay particles with intercalation chemistry. The presence of the nanosilicates increases the Young's modulus and stalls the degradation rate of the resulting hydrogels. We demonstrate that the reinforced hydrogels promote the proliferation as well as the attachment and induced the differentiation of encapsulated mesenchymal stem cells in vitro. Furthermore, we explore the effects of nanoengineered hydrogels in vivo with the critical-sized mouse calvarial defect model. Our results confirm that chitosan-montmorillonite hydrogels are able to recruit native cells and promote calvarial healing without delivery of additional therapeutic agents or stem cells, indicating their tissue engineering potential.

Cartilage tissue regeneration often presents a challenging clinical situation. Recently, it has been shown that Periodontal Ligament Stem Cells (PDLSCs) possess high chondrogenic differentiation capacity. In this study, we developed a stem cell delivery system based on alginate/hyaluronic acid (HA) loaded with TGF-β1 ligand, encapsulating PDLSCs; and investigated the chondrogenic differentiation of encapsulated cells in alginate/HA hydrogel microspheres in vitro and in vivo. The results showed that PDLSCs, as well as human bone marrow mesenchymal stem cells (hBMMSCs), as the positive control, were stained positive for both toluidine blue and alcian blue staining, while exhibiting high levels of gene expression related to chondrogenesis (Col II, Aggrecan and Sox-9), as assessed via qPCR. The quantitative PCR analyses exhibited that the chondrogenic differentiation of encapsulated MSCs can be regulated by the modulus of elasticity of hydrogel delivery system, confirming the vital role of the microenvironment, and the presence of inductive signals for viability and differentiation of MSCs. In vivo, histological and immunofluorescence staining for chondrogenic specific protein markers confirmed ectopic cartilage-like tissue regeneration inside transplanted hydrogels. PDLSCs presented significantly greater capability for chondrogenic differentiation than hBMMSCs (P < 0.05). Altogether, our findings confirmed that alginate/HA hydrogels encapsulating PDLSCs are a promising candidate for cartilage regeneration.

Mesoderm derived from human embryonic stem cells (hESCs) is a major source of the mesenchymal stem/stromal cells (MSCs) that can differentiate into osteoblasts and chondrocytes for tissue regeneration. While significant progress has been made in understanding of molecular mechanisms of hESC differentiation into mesodermal cells, little is known about epigenetic factors controlling hESC fate toward mesoderm and MSCs. Identifying potential epigenetic factors that control hESC differentiation will undoubtedly lead to advancements in regenerative medicine. Here, we conducted an epigenome-wide analysis of hESCs and MSCs and uncovered that EZH2 was enriched in hESCs and was downregulated significantly in MSCs. The specific EZH2 inhibitor GSK126 directed hESC differentiation toward mesoderm and generated more MSCs by reducing H3K27me3. Our results provide insights into epigenetic landscapes of hESCs and MSCs and suggest that inhibiting EZH2 promotes mesodermal differentiation of hESCs., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy., Statement of Significance: Application of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Accurate and early prediction of tissue viability is the most significant determinant of tissue flap survival in reconstructive surgery. Perturbation in tissue water content (TWC) is a generic component of the tissue response to such surgeries, and, therefore, may be an important diagnostic target for assessing the extent of flap viability in vivo . We have previously shown that reflective terahertz (THz) imaging, a non-ionizing technique, can generate spatially resolved maps of TWC in superficial soft tissues, such as cornea and wounds, on the order of minutes. Herein, we report the first in vivo pilot study to investigate the utility of reflective THz TWC imaging for early assessment of skin flap viability. We obtained longitudinal visible and reflective THz imagery comparing 3 bipedicled flaps (i.e. survival model) and 3 fully excised flaps (i.e. failure model) in the dorsal skin of rats over a postoperative period of 7 days. While visual differences between both models manifested 48 hr after surgery, statistically significant ( p < 0.05, independent t -test) local differences in TWC contrast were evident in THz flap image sets as early as 24 hr. Excised flaps, histologically confirmed as necrotic, demonstrated a significant, yet localized, reduction in TWC in the flap region compared to non-traumatized skin. In contrast, bipedicled flaps, histologically verified as viable, displayed mostly uniform, unperturbed TWC across the flap tissue. These results indicate the practical potential of THz TWC sensing to accurately predict flap failure 24 hours earlier than clinical examination.

Pluripotent or multipotent cell-based therapeutics are vital for skeletal reconstruction in non-healing critical-sized defects since the local endogenous progenitor cells are not often adequate to restore tissue continuity or function. However, currently available cell-based regenerative strategies are hindered by numerous obstacles including inadequate cell availability, painful and invasive cell-harvesting procedures, and tumorigenesis. Previously, we established a novel platform technology for inducing a quiescent stem cell-like stage using only a single extracellular proteoglycan, fibromodulin (FMOD), circumventing gene transduction. In this study, we further purified and significantly increased the reprogramming rate of the yield multipotent FMOD reprogrammed (FReP) cells. We also exposed the 'molecular blueprint' of FReP cell osteogenic differentiation by gene profiling. Radiographic analysis showed that implantation of FReP cells into a critical-sized SCID mouse calvarial defect, contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario in vivo. The persistence, engraftment, and osteogenesis of transplanted FReP cells without tumorigenesis in vivo were confirmed by histological and immunohistochemical staining. Taken together, we have provided an extended potency, safety, and molecular profile of FReP cell-based bone regeneration. Therefore, FReP cells present a high potential for cellular and gene therapy products for bone regeneration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A high-fat (HF) diet inducing hyperlipidemia has been associated with the pathophysiology of major diseases, such as atherosclerosis and osteoporosis. A HF diet has significant adverse effects on bone, including lower bone density, volume, and strength. Statins, drugs that lower serum cholesterol levels have beneficial effects on bone metabolism. Since the host's bone quantity, quality, and healing potential play a crucial role in osseointegration of dental implants, we hypothesized that hyperlipidemia may negatively affect implant osseointegration. In the present study, we evaluated the effects of hyperlipidemia on implant osseointegration in mice. Atherosclerosis susceptible C57BL/6J male mice were randomly placed on a control chow or a HF diet. After 12 weeks on the diet, each mouse received a titanium implant in the proximal metaphysis of the femur. The animals were humanely killed at 4 or 8 weeks after the implant surgery. Results showed that the mice fed a HF diet had significantly increased implant loss as well as decreased formation and strength of bone-to-implant interface. These results support the hypothesis that a HF diet can significantly compromise osseointegration, causing poor outcome in dental implant therapy.

Hydrogels have a unique opportunity to regenerate damaged cartilage tissues by introducing mesenchymal stem cells (MSCs) in a highly swollen environment similar to articular cartilage. During cartilage development, collagen-cell interactions play an important role in mediating early mesenchymal condensation and chondrogenesis with transforming growth factor-β1 (TGF-β1) stimulation. Here, a hydrogel environment that can enhance cell-matrix interactions and chondrogenesis by stabilizing type-II collagen (Col II) and TGF-β1 into photopolymerizable (methacrylated) chitosan (MeGC) with simple entrapment and affinity binding is demonstrated. The MeGC hydrogel was designed to gel upon initiation by exposure to visible blue light in the presence of riboflavin, an aqueous initiator from natural vitamin. The incorporation of Col II into MeGC hydrogels increased cellular condensation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. MeGC hydrogels containing Col II supported the release of TGF-β1 in a controlled manner over time in chondrogenic medium and the incorporated TGF-β1 further enhanced chondrogenesis of encapsulated chondrocytes and MSCs, especially synovial MSCs. Subcutaneous implantation of hydrogel cultures showed greatly improved neocartilage formation in constructs loaded with TGF-β1 compared with controls. These findings suggest that cartilage mimetic hydrogels have a high potential for cartilage repair., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Bisphosphonates (BPs) are anti-resorptive agents commonly used to treat bone-related diseases; however, soft tissue-related side-effects are frequently reported in some BP users, such as oral or gastrointestinal (GI) ulcerations. BPs are stable analogs of pyrophosphate and have high affinity to hydroxyapatite, allowing them to bind to the bone surfaces and exert suppressive effects on osteoclast functions. However, the underlying mechanisms as to how bone-seeking BPs also exert cytotoxic effects on soft tissue remain unknown. In the present study, we investigated the localization of nitrogen-containing BPs (N-BPs) in hard and soft tissue using fluorescently-labeled N-BPs in vitro. We developed osteomucosal tissue constructs in vitro to recapitulate the hard and soft tissue of the oral cavity. A histological examination of the osteomucosal tissue constructs revealed a differentiated epithelium over the bone containing osteocytes and the periosteum, similar to that observed in the rat palatal tissues. Following treatment with the fluorescently-labeled bisphosphonate, AF647-ZOL, the osteomucosal constructs exhibited fluorescent signals, not only in the bone, but also in the epithelium. No fluorescent signals were observed from the control- or ZOL-treated constructs, as expected. Collectively, the data from the present study suggest that N-BPs localize to epithelial tissue and that such a localization and subsequent toxicity of N-BPs may be associated, at least in part, with soft tissue-related side-effects.

Hyperlipidemia increases the risk for generation of lipid oxidation products, which accumulate in the subendothelial spaces of vasculature and bone. Atherogenic high-fat diets increase serum levels of oxidized lipids, which are known to attenuate osteogenesis in culture and to promote bone loss in mice. In this study, we investigated whether oxidized lipids affect bone regeneration and mechanical strength. Wild-type (WT) and hyperlipidemic (Ldlr(-/-)) mice were placed on a high-fat (HF) diet for 13 weeks. Bilateral cranial defects were introduced on each side of the sagittal suture, and 5 weeks postsurgery on the respective diets, the repair/regeneration of cranial bones and mechanical properties of femoral bones were assessed. MicroCT and histological analyses demonstrated that bone regeneration was significantly impaired by the HF diet in WT and Ldlr(-/-) mice. In femoral bone, cortical bone volume fraction (bone volume [BV]/tissue volume [TV]) was significantly reduced, whereas cortical porosity was increased by the HF diet in Ldlr(-/-) but not in WT mice. Femoral bone strength and stiffness, measured by three-point bending analysis, were significantly reduced by the HF diet in Ldlr(-/-), but not in WT mice. Serum analysis showed that the HF diet significantly increased levels of parathyroid hormone, tumor necrosis factor (TNF)-α, calcium, and phosphorus, whereas it reduced procollagen type I N-terminal propeptide, a serum marker of bone formation, in Ldlr(-/-), but not in WT mice. The serum level of carboxyl-terminal collagen crosslinks, a marker for bone resorption, was also 1.7-fold greater in Ldlr(-/-) mice. These findings suggest that hyperlipidemia induces secondary hyperparathyroidism and impairs bone regeneration and mechanical strength.

Nell-1 is a recently discovered secreted protein with the capacity to promote osteoblastic calvarial cell differentiation and mineralization and induce calvarial bone overgrowth and regeneration in various rodent models. However, the extent of Nell-1 osteoinductivity in large animal cells remains unknown. The objective of the study was to evaluate the feasibility of adenoviral encoding Nell-1 (AdNell-1) gene transfer into primary adult goat bone marrow stromal cells (BMSCs) in vitro and in vivo and to compare the osteoinductive effects with those produced by bone morphogenetic protein-2 (BMP-2), a well established osteoinductive molecule currently utilized for regional gene therapy. AdNell-1-transduced BMSCs expressed Nell-1 protein and underwent osteoblastic differentiation within 2 weeks in vitro, which is comparable to AdBMP-2. After intramuscular injection of nude mice, the AdNell-1- and AdBMP-2-transduced BMSCs revealed new bone formation, while untransduced or AdLacZ-transduced BMSCs showed mainly fibrotic tissue proliferation. At 4 weeks, BMP-2 induced significantly larger bone mass with a mature bone margin and central cavity filled with primarily fatty marrow tissue. Nell-1 samples had significantly less bone mass but were histologically similar to newly formed trabecular bone mixed with chondroid bone-like areas verified by type X collagen (ColX) immunohistochemistry. This distinct difference in histomorphology from the bone mass induced by BMP-2 suggests that there is a potential clinical role/advantage for Nell-1 in skeletal tissue engineering and regeneration.

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-beta1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration.