Genetic and pharmacologic suppression of PPARγ enhances NELL-1-stimulated bone regeneration.

Recent investigations into mechanisms behind the development of osteoporosis suggest that suppressing PPARγ-mediated adipogenesis can improve bone formation and bone mineral density. In this study, we investigated a co-treatment strategy to enhance bone formation by combining NELL-1, an osteogenic molecule that has been extensively studied for its potential use as a therapeutic for osteoporosis, with two methods of PPARγ suppression. First, we suppressed PPARγ genetically using lentiviral PPARγ-shRNA in immunocompromised mice for a proof of concept. Second, we used a PPARγ antagonist to suppress PPARγ pharmacologically in immunocompetent senile osteopenic mice for clinical transability. We found that the co-treatment strategy significantly increased bone formation, increased the proliferation stage cell population, decreased late apoptosis of primary mouse BMSCs, and increased osteogenic marker mRNA levels in comparison to the single agent treatment groups. The addition of PPARγ suppression to NELL-1 therapy enhanced NELL-1's effects on bone formation by upregulating anabolic processes without altering NELL-1's inhibitory effects on osteoclastic and adipogenic activities. Our findings suggest that combining PPARγ suppression with therapeutic NELL-1 may be a viable method that can be further developed as a novel strategy to reverse bone loss and decrease marrow adiposity in age-related osteoporosis.

NELL-1 injection maintains long-bone quantity and quality in an ovariectomy-induced osteoporotic senile rat model.

Over 10 million Americans have osteoporosis, and is the predominant cause of fractures in the elderly. Treatment of fractures in the setting of osteoporosis is complicated by a suboptimal bone regenerative response due to a decline in the number of osteoblasts, their function, and survival. Consequently, an osteogenic therapeutic to prevent and treat fractures in patients with osteoporosis is needed. Nel-like molecule-1 (NELL-1), a novel osteoinductive growth factor, has been shown to promote bone regeneration. In this study, we aim to demonstrate the capacity of recombinant NELL-1 to prevent ovariectomy (OVX)-induced osteoporosis in a senile rat model. Ten-month-old female Sprague-Dawley rats underwent either sham surgery or OVX. Subsequently, 50 µL of 600 µg/mL NELL-1 lyophilized onto a 0-50-µm tricalcium phosphate (TCP) carrier was injected into the femoral bone marrow cavity while phosphate-buffered saline (PBS) control was injected into the contralateral femur. Our microcomputed tomography results showed that OVX+PBS/TCP control femurs showed a continuous decrease in the bone volume (BV) and bone mineral density (BMD) from 2 to 8 weeks post-OVX. In contrast, OVX+NELL-1/TCP femurs showed resistance to OVX-induced bone resorption showing BV and BMD levels similar to that of SHAM femurs at 8 weeks post-OVX. Histology showed increased endosteal-woven bone, as well as decreased adipocytes in the bone marrow of NELL-1-treated femurs compared to control. NELL-1-treated femurs also showed increased immunostaining for bone differentiation markers osteopontin and osteocalcin. These findings were validated in vitro, in which addition of NELL-1 in OVX bone marrow stem cells resulted in increased osteogenic differentiation. Thus, NELL-1 effectively enhances in situ osteogenesis in the bone marrow, making it potentially useful in the prevention and treatment of osteoporotic fractures.

NELL-1 promotes cartilage regeneration in an in vivo rabbit model.

Repair of cartilage due to joint trauma remains challenging due to the poor healing capacity of cartilage and adverse effects related to current growth factor-based strategies. NELL-1 (Nel-like molecule-1; Nel [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]), a protein first characterized in the context of premature cranial suture fusion, is believed to accelerate differentiation along the osteochondral lineage. We previously demonstrated the ability of NELL-1 protein to maintain the cartilaginous phenotype of explanted rabbit chondrocytes in vitro. Our objective in the current study is to determine whether NELL-1 can affect endogenous chondrocytes in an in vivo cartilage defect model. To generate the implant, NELL-1 was incorporated into chitosan nanoparticles and embedded into alginate hydrogels. These implants were press fit into 3-mm circular osteochondral defects created in the femoral condylar cartilage of 3-month-old New Zealand White rabbits (n=10). Controls included unfilled defects (n=8) and defects filled with phosphate-buffered saline-loaded chitosan nanoparticles embedded in alginate hydrogels (n=8). Rabbits were sacrificed 3 months postimplantation for histological analysis. Defects filled with alginate containing NELL-1 demonstrated significantly improved cartilage regeneration. Remarkably, histology of NELL-1-treated defects closely resembled that of native cartilage, including stronger Alcian blue and Safranin-O staining and increased deposition of type II collagen and absence of the bone markers type I collagen and Runt-related transcription factor 2 (Runx2) as demonstrated by immunohistochemistry. Our results suggest that NELL-1 may produce functional cartilage with properties similar to native cartilage, and is an exciting candidate for tissue engineering-based approaches for treating diverse pathologies of cartilage defects and degeneration.

The Nell-1 growth factor stimulates bone formation by purified human perivascular cells.

The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation.

Stability comparison between commercially available mini-implants and a novel design: part 1.

OBJECTIVE: To compare mechanical stability among five mini-implant designs--a newly invented design and four commercially available designs that vary by shape and threading; to calculate external surface area of each design using high-resolution micro-computed tomography; and to evaluate the relationship between surface area and stability results. MATERIALS AND METHODS: The four commercially available mini-implants--single-threaded and cylindrical (SC), single-threaded and tapered (ST), double-threaded and cylindrical (DC), double-threaded and tapered (DT)--and a new implant that is designed to engage mostly in cortical bone with shorter and wider dimensions (N1) were inserted in simulated bone with cortical and trabecular bone layers. The mechanical study consisted of torque measurements and lateral displacement tests. External surface area was computed using a 25-µm micro-CT. RESULTS: Maximum insertion torque, maximum removal torque, and force levels for displacements were the highest in N1, followed by DT, ST, DC, and SC (α  =  .05). The surface area was largest in DT, followed by N1, ST, DC, and SC. Surface area engaged in cortical bone, however, was the greatest in N1. The surface area of mini-implants had positive correlation with stability. CONCLUSION: Among commercial designs, both added tapering and double threading improved stability. N1 was the most stable design within this research design. The new design has the potential to be clinically superior; it has enhanced stability and there is diminished risk of endangering nearby anatomic structures during placement and orthodontic treatment, but the design requires refinements to reduce insertion torque to avoid clinical difficulty and patient discomfort.

High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.

The major Food and Drug Association-approved osteoinductive factors in wide clinical use are bone morphogenetic proteins (BMPs). Although BMPs can promote robust bone formation, they also induce adverse clinical effects, including cyst-like bone formation and significant soft tissue swelling. In this study, we evaluated multiple BMP2 doses in a rat femoral segmental defect model and in a minimally traumatic rat femoral onlay model to determine its dose-dependent effects. Results of our femoral segmental defect model established a low BMP2 concentration range (5 and 10 µg/mL, total dose 0.375 and 0.75 µg in 75 µg total volume) unable to induce defect fusion, a mid-range BMP2 concentration range able to fuse the defect without adverse effects (30 µg/mL, total dose 2.25 µg in 75 µg total volume), and a high BMP2 concentration range (150, 300, and 600 µg/mL, total dose 11.25, 22.5, and 45 µg in 75 µg total volume) able to fuse the defect, but with formation of cyst-like bony shells filled with histologically confirmed adipose tissue. In addition, compared to control, 4 mg/mL BMP2 also induced significant tissue inflammatory infiltrates and exudates in the femoral onlay model that was accompanied by increased numbers of osteoclast-like cells at 3, 7, and 14 days. Overall, we consistently reproduced BMP2 side effects of cyst-like bone and soft tissue swelling using high BMP2 concentration approaching the typical human 1500 µg/mL.

Long-term stability of anterior open-bite treatment by intrusion of maxillary posterior teeth.

INTRODUCTION: Anterior open bite results from the combined influences of skeletal, dental, functional, and habitual factors. The long-term stability of anterior open bite corrected with absolute anchorage has not been thoroughly investigated. The purpose of this study was to examine the long-term stability of anterior open-bite correction with intrusion of the maxillary posterior teeth. METHODS: Nine adults with anterior open bite were treated by intrusion of the maxillary posterior teeth. Lateral cephalographs were taken immediately before and after treatment, 1 year posttreatment, and 3 years posttreatment to evaluate the postintrusion stability of the maxillary posterior teeth. RESULTS: On average, the maxillary first molars were intruded by 2.39 mm (P<0.01) during treatment and erupted by 0.45 mm (P<0.05) at the 3-year follow-up, for a relapse rate of 22.88%. Eighty percent of the total relapse of the intruded maxillary first molars occurred during the first year of retention. Incisal overbite increased by a mean of 5.56 mm (P<0.001) during treatment and decreased by a mean of 1.20 mm (P<0.05) by the end of the 3-year follow-up period, for a relapse rate of 17.00%. Incisal overbite significantly relapsed during the first year of retention (P<0.05) but did not exhibit significant recurrence between the 1-year and 3-year follow-ups. CONCLUSIONS: Most relapse occurred during the first year of retention. Thus, it is reasonable to conclude that the application of an appropriate retention method during this period clearly enhances the long-term stability of the treatment.

The use of BMP-2 coupled - Nanosilver-PLGA composite grafts to induce bone repair in grossly infected segmental defects.

Healing of contaminated/infected bone defects is a significant clinical challenge. Prevalence of multi-antibiotic resistant organisms has renewed interest in the use of antiseptic silver as an effective, but less toxic antimicrobial with decreased potential for bacterial resistance. In this study, we demonstrated that metallic nanosilver particles (with a size of 20-40nm)-poly(lactic-co-glycolic acid) (PLGA) composite grafts have strong antibacterial properties. In addition, nanosilver particles-PLGA composite grafts did not inhibit adherence, proliferation, alkaline phosphatase activity, or mineralization of ongrowth MC3T3-E1 pre-osteoblasts compared to PLGA controls. Furthermore, nanosilver particles did not affect the osteoinductivity of bone morphogenetic protein 2 (BMP-2). Infected femoral defects implanted with BMP-2 coupled 2.0% nanosilver particles-PLGA composite grafts healed in 12 weeks without evidence of residual bacteria. In contrast, BMP-2 coupled PLGA control grafts failed to heal in the presence of continued bacterial colonies. Our results indicate that nanosilver of defined particle size is bactericidal without discernable in vitro and in vivo cytotoxicity or negative effects on BMP-2 osteoinductivity, making it an ideal antimicrobial for bone regeneration in infected wounds.

Topographic distribution area of the infraorbital nerve.

The infraorbital nerve (ION) supplies the skin and mucous membranes of the middle portion of the face. This nerve is vulnerable to injury during surgical procedures of mid-face. Severe pain and loss of sense are noted in patients whose infraorbital nerve is either entirely or partially lost after these surgeries. We investigated the distribution area and topography of the ION, about which little is currently known, by dissecting 43 hemifaces of Korean cadavers. The ION produced four main branches, the inferior palpebral, internal nasal, external nasal, and superior labial branches. The inferior palpebral branch was generally bifurcated, giving off a medial and a lateral branch (58.1%). The internal nasal branch ran superior to the depressor septi muscle, along the ala of the nose. It supplied the skin of the philtrum and gave off a terminal branch that supplied the nasal septum and the vestibule of the nose. The external nasal branch was distributed diversely supplying areas between the root and the ala of the nose. The superior labial branch was the largest branch of the ION produced the most subbranches. These subbranches were divided into the medial and lateral branches depending upon the area that they supplied.